Vaccines and diagnostics for the ehrlichioses

ABSTRACT

The present invention concerns VLPT immunoreactive compositions for  E. chaffeensis  and compositions related thereto, including vaccines, antibodies, polypeptides, peptides, and polynucleotides. In particular, epitopes for  E. chaffeensis  VLPT are disclosed.

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2009/030527 filed Jan. 9, 2009 whichclaims priority to U.S. Provisional Patent Application Ser. No.61/020,379, filed Jan. 10, 2008, which is incorporated by referenceherein in its entirety.

The present invention was made at least in part by funds from theNational Institutes of Health grant R01AI 071145-01. The United StatesGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention concerns at least the fields of molecular biology,cell biology, pathology, and medicine, including veterinary medicine. Inspecific aspects, the present invention concerns immunoreactive VLPTcompositions in E. chaffeensis.

BACKGROUND OF THE INVENTION

Ehrlichia chaffeensis is a tick-transmitted obligately intracellularbacterium that causes human monocytrotropic ehrlichiosis (HME), anemerging life-threatening disease in humans and also causes mild tosevere disease in wild and domestic canids. (Paddock and Childs, 2003).The genomes of E. canis and other organisms in the genus, including E.chaffeensis and E. ruminantium, exhibit a high degree of genomicsynteny, paralogous protein families, a large proportion of proteinswith transmembrane helices and/or signal sequences, and a uniqueserine-threonine bias associated with potential for O-glycosylation andphosphorylation, and have tandem repeats and ankyrin domains in proteinsassociated with host-pathogen interactions (Collins et al., 2005;Dunning Hotopp et al., 2006; Frutos et al., 2006; Mavromatis et al.,2006). A small subset of the more than 900 proteins encoded by each ofthese genomes are recognized by antibody (Doyle et al., 2006; McBride etal., 2003; McBride et al., 2000). Several of the major immunoreactiveproteins identified and molecularly characterized are serine-richglycoproteins that are secreted. Many of these glycoproteins have tandemrepeats; however, one has numerous eukaryote-like ankyrin domains (Doyleet al., 2006; McBride et al., 2003; McBride et al., 2000; Nethery etal., 2005; Singu et al., 2005; Yu et al., 2000).

Three immunoreactive proteins with tandem repeats have been identifiedand molecularly characterized in E. chaffeensis (gp120, gp47, and VLPT)as well as two orthologs in E. canis (gp140 and gp36, respectively). E.chaffeensis gp120 and gp47 are major immunoreactive proteins that aredifferentially expressed on the surface dense-cored ehrlichiae and aresecreted (Doyle et al., 2006; Popov, et al. 2000). Extensive variabilityin the number and/or sequence of tandem repeats in the E. chaffeensisimmunoreactive proteins (gp120, gp47 and VLPT) as well as E. canis gp36is well documented (Chen et al., 1997; Doyle et al., 2006; Sumner etal., 1999). The gp120 contains two to five nearly identical serine-richTRs with 80-amino acids each, and gp47 has carboxy-terminal serine-richTRs that vary in number and amino acid sequence among different isolatesof each species. Major antibody epitopes of both gp120 and gp47 havebeen mapped to these serine-rich acidic TRs. (Doyle et al., 2006; Yu etal. 1996; Yu et al. 1997). Similarly, the VLPT has three to sixnonidentical serine-rich TRs (30 amino acids); however, the E. canisortholog (gp19) lacks multiple TRs. The presence of tandem repeats inboth coding and noncoding regions of the genome has been linked to anactive process of expansion and reduction of ehrlichial genomes (Frutoset al., 2006) and is considered a major source of genomic change andinstability (Bzymek and Lovett, 2001). The E. chaffeensis vlpt gene alsoexhibits variations in the number of 90-bp tandem repeats (2 to 6) andhas been utilized as a molecular diagnostic target and fordifferentiation of isolates (Sumner et al., 1999; Yabsley et al., 2003).

Recently, a strongly acidic 19-kDa major immunoreactive protein of E.canis has been identified (gp19), having the same relative chromosomallocation and substantial homology in a C-terminal cysteine-tyrosine-richdomain as previously reported for VLPT protein in E. chaffeensis.However, while E. chaffeensis VLPT is immunoreactive, little is knownregarding its cellular location, function and role in development ofprotective immunity. The molecular mass of native VLPT is also unknown.It has been suggested that E. chaffeensis Arkansas strain was 44-kDa,but immunoreactive proteins consistent with that mass have not beenreported (Sumner et al. 1999). The VLPT of E. chaffeensis Arkansas is a198 amino acid protein that has four repeats (30 amino acids) and has amolecular mass approximately double that predicted by its amino acidsequence (Sumner et al., 1999). E. chaffeensis VLPT protein appears tohave posttranslational modification consistent with other describedehrlichial glycoproteins, but the presence of carbohydrate on VLPT hasnot been demonstrated.

Defining the molecular characteristics of ehrlichial immunodeterminantsinvolved in eliciting humoral immunity during infection is important forunderstanding the basis of immunity to Ehrlichia species. The presentinvention fulfills a need in the art by providing novel methods andcompositions concerning erhlichial infections in mammals, and inparticular provides methods and compositions utilizing E. chaffeensisVLPT.

SUMMARY OF THE INVENTION

Human monocytrotropic ehrlichiosis (HME) is a tick-borne disease causedby the obligate intracellular bacterium Ehrlichia chaffeensis. Ingeneral, the present invention concerns ehrlichial compositions andmethods of manufacturing and using them. In specific embodiments, theinvention concerns immunogenic compositions, including, for example,immunoprotective antigens as vaccines for ehrlichial diseases, such assubunit vaccines, for example. The immunogenic composition may beemployed for any mammal, including, for example, humans, dogs, cats,horses, pigs, goats, or sheep.

Ehrlichia chaffeensis and E. canis have a small subset of tandem-repeat(TR) containing proteins that elicit strong host immune responses andare associated with host-pathogen interactions. Previously, a highlyconserved 19-kDa major immunoreactive protein (gp19) of E. canis wascharacterized and the corresponding TR-containing orthologvariable-length PCR target (VLPT) protein in E. chaffeensis wasidentified. In an embodiment of this invention, the native 32-kDa VLPTprotein is identified and the immunodeterminants defined in order tofurther define the molecular basis of the host immune response to E.chaffeensis. Synthetic and/or recombinant polypeptides corresponding tovarious regions of VLPT were used to localize major antibody epitopes tothe TR-containing region. Major antibody epitopes were identified inthree non-identical repeats (R2, R3 and R4), which reacted strongly withantibodies in sera from an E. chaffeensis-infected dog and HME patients.VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitopewas further localized to a nearly identical proximal 17-amino-acidregion common between these repeats that was species-specific. Theepitope in R4 was distinct from that of R2 and R3 and was found to haveconformational dependence. VLPT was detected in supernatants frominfected cells, indicating that the protein was secreted. VLPT waslocalized on both reticulate and dense-cored cells, and it was foundextracellularly in the morula fibrillar matrix and associated with themorula membrane.

In certain aspects of the invention, there is identification andcharacterization of the major immunoreactive glycoprotein VLPT in E.chaffeensis, the ortholog of the E. canis gp19. The E. canis gp19 lackstandem repeats present in VLPT of E. chaffeensis, but the two proteinsexhibit substantial amino acid similarity (59%) in acysteine/tyrosine-rich carboxyl-terminal region, and both genes have thesame relative chromosomal location. It was found that carbohydrate onrecombinant ehrlichial TR-containing proteins exhibited larger thanpredicted masses similar to their native counterparts. VLPT exhibits alarger than predicted mass by gel electrophoresis, a finding that isobserved with both native and recombinant VLPT proteins. Serine andthreonine residues are linkage sites for O-glycans, and some of theseamino acids were predicted to be glycan attachment sites on the VLPT.However, unlike other ehrlichial proteins, carbohydrate was not found onthe VLPT, and the mass (as determined by MALDI-TOF) of a recombinant tworepeat containing fragment (VLPT-R32; the combination of repeats R3 andR2) was consistent with its predicted mass confirming that the abnormalmigration was not due to post-translational modification of VLPT tandemrepeats. In an alternative embodiment, however, the VLPT ispost-translationally modified. VLPT is a highly acidic protein, which incertain embodiments relates to the increase in electrophoretic mobility.In specific embodiments, the high acidic amino acid content and lowoverall pI (3.8) of VLPT relates to its electrophoretic behavior and, inparticular cases contributes to the anomalous behavior of other highlyacidic TR-containing ehrlichial proteins.

In specific aspects of the present invention, there are ehrlichial VLPTpolypeptide compositions (or polynucleotide compositions that encode allor part of them) with one or more of the following characteristics: 1)comprises one or more moieties, which in specific embodiments comprisespart of an epitope determinant; 2) comprises one or more moieties, suchas an epitope, that are immunogenically species-specific; 3) is releasedextracellularly, such as by secretion; 4) comprises major B cellepitopes; 5) is surface-exposed; 6) is associated with the infectiousdense-cored forms of ehrlichiae, such as on the surface, for example;and 7) is associated with morula fibrils (ehrlichiae form microcoloniesinside cellular vacuoles (morulae) that harbor many individualehrlichiae). In further aspects, recombinant polypeptide compositions ofthe present invention may be employed as an immunogenic composition,including, for example, a vaccine.

In particular embodiments of the invention, there are E. chaffeensisVLPT immunogenic compositions that comprise an amino acid sequence thatis immunogenic, and in further particular embodiments, theimmunogenicity is characterized by being at least part of an epitope. Infurther embodiments, the amino acid sequence comprises at least part ofa vaccine composition against an ehrlichial organism, such as E.chaffeensis. In specific embodiments, the amino acid sequence comprisesserines, threonines, and/or, optionally, alanine, proline, valine,and/or glutamic acid.

In further specific embodiments, an amino acid sequence of theinvention, for example an immunogenic amino acid sequence, comprisespart or all of the following exemplary sequence:SDSHEPSHLELPSLSEEVIQLESDLQQSSN (SEQ ID NO:3); exemplary sequence:SDLHGSFSVELFDPFKEAVQLGNDLQQSSD (SEQ ID NO:4); exemplary sequence:SDLHGSFSVELFDPSKEEVQLESDLQQSSN (SEQ ID NO:5); exemplary sequence:SDLHGSFSVELFDPFKE (SEQ ID NO:8) exemplary sequence: HGSFSVELFDPFKE (SEQID NO:9); exemplary sequence: HGSFSVELFDPFKEAVQ (SEQ ID NO:10); orexemplary sequence: HGSFSVELFDPFKEAVQLGN (SEQ ID NO:11). In additionalembodiments, the amino acid sequence is comprised in a pharmaceuticallyacceptable excipient, which in some aspects of the invention comprisesan adjuvant. In certain aspects of the invention, there is apolynucleotide comprising SEQ ID NO:16(tttatatttatatatgattaatatataatgataatggtatgtggttataactgcttattagttgatcatgtacctgtgtgttatgttaaatagggtataaatatgtcacaattctctgaagataatatgggtaatatacaaatgccttttgattctgattcacatgagccttctcatcttgagctacctagtctttctgaagaagtgattcaattagagagtgatctacaacaatcttctaattctgatttacacgggtctttttctgttgagttatttgatccttttaaagaagcagttcaattggggaatgatctacaacaatcttctgattctgatttacacgggtctttttctgttgagttatttgatccttctaaagaagaagttcaattggagagtgatctacaacaatcttctaattctgatttacacgagtcttcttttgttgagttacctggtccttccaaagaagaagttcaattcgaagatgatgctaaaaatgtagtatatggacaagaccatgttagtttatctgaattaggcttattgttaggtggtgtttttagtacaatgaattatttgtctggttatacaccgtattattatcatcattattgttgttataatccttattattattttgattatgttactccagattattgtcatcactgtagtgaaagtagtttagagtaggatatttagaaatataaatggttgttgacttcacaaaaggtgtagttttatatgttttatgctgttttatagtgttataaggatatgagttgtttttactattttt) that encodes the peptide sequence of SEQID NO:1 (MSQFSEDNMGNIQMPFDSDSHEPSHLELPSLSEEVIQLESDLQQSSNSDLHGSFSVELFDPFKEAVQLGNDLQQSSDSDLHGSFSVELFDPSKEEVQLESDLQQSSNSDLHESSFVELPGPSKEEVQFEDDAKNVVYGQDHVSLSELGLLLGGVFSTMNYLSGYTPYYYHHYCCYNPYYYFDYVTPDYCHHCSESSLE).

In certain embodiments of the present invention, there are immunogenicVLPT E. chaffeensis compositions, and particular sequences of the VLPTcompositions may impart its immunogenicity; for example, a region of theVLPT composition may comprise an epitope.

In some aspects of the invention, multiple different E. chaffeensisstrains comprise immunogenic VLPT compositions, and there is significantsequence identity among the strains in regions of the VLPT compositionsthat comprise the epitope (such as greater than about 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example). However,in some embodiments, there may be significant sequence identity amongthe strains in regions of the VLPT compositions that do not comprise theepitope. In particular aspects of the invention, there is a VLPTcomposition that is immunogenic for more than one strain of E.chaffeensis, and in particular aspects, the epitope of one of thestrains is or comprises or consists essentially of or consists of SEQ IDNO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10;or SEQ ID NO:11, although other epitopes may also be identified. Inembodiments wherein an alternative VLPT E. chaffeensis epitope isidentified, there may be provided an immunogenic composition comprisinga mixture of VLPT E. chaffeensis epitopes, such as a mixture includingSEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ IDNO:10; or SEQ ID NO:11, for example.

In an embodiment of the invention, there is an immunogenic VLPT E.chaffeensis glycoprotein. In an additional embodiment of the invention,there is an E. chaffeensis composition comprising SEQ ID NO:3; SEQ IDNO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ IDNO:11. In specific aspects of the invention, the composition furthercomprises a pharmaceutically acceptable excipient. The composition maybe further defined as comprising one or more carbohydrate moieties, ascomprising part or all of an epitope, and/or as a vaccine, such as asubunit vaccine.

In another embodiment of the invention, there is an E. chaffeensiscomposition comprising a polypeptide encoded by at least part of thepolynucleotide of SEQ ID NO:16 and/or an E. chaffeensis compositioncomprising a polypeptide of SEQ ID NO:1. In one embodiment of theinvention, there is an isolated composition comprising an Ehrlichia VLPTglycoprotein, comprising: (a) a sequence selected from the groupconsisting of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ IDNO:9; SEQ ID NO:10; or SEQ ID NO:11; or (b) a sequence that is at leastabout 70% identical to one or more sequences in (a). The composition maybe further defined as a sequence that is at least about 75%, about 80%,about 85%, about 90%, or about 95% identical to one or more sequences in(a). The composition may also be further defined as being comprised in apharmaceutically acceptable excipient, as comprising one or morecarbohydrate moieties, and/or as being comprised in a pharmaceuticalcomposition suitable as a vaccine.

In a specific embodiment, there is an isolated polynucleotide thatencodes SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9;SEQ ID NO:10; or SEQ ID NO:11, or a mixture thereof.

In particular embodiments, there is an isolated polynucleotide,comprising: a) a polynucleotide that encodes SEQ ID NO:3; SEQ ID NO:4;SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11; orb) a polynucleotide that is at least about 90% identical to thepolynucleotide of a) and that encodes an immunoreactive E. chaffeensisVLPT polypeptide.

In an additional embodiment of the invention, there is an isolatedpolypeptide, comprising: a) SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11, or b) a VLPTpolypeptide that is at least about 70% identical to SEQ ID NO:3; SEQ IDNO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ IDNO:11, and that comprises immunogenic activity. In a specificembodiment, the polypeptide is comprised in a pharmaceuticallyacceptable excipient, and/or it may be further defined as beingcomprised in a pharmaceutical composition suitable as a vaccine.

In certain aspects of the invention, there are polynucleotides that areamplifiable by one or more of the exemplary primers of SEQ ID NO:17, SEQID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22(Table 4.)

In another aspect of the invention, there are isolated antibodies thatbind one or more polypeptides of the invention. Antibodies may bemonoclonal, polyclonal, or antibody fragments, for example. Inparticular embodiments, the antibody binds selectively to an epitope ofVLPT, for example one that comprises SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11. Inspecific embodiments, the antibody may be referred to as immunologicallyreacting with one or more polypeptides of the invention.

In an additional embodiment of the invention, there is a method ofproviding resistance to E. chaffeensis infection, comprising the step ofdelivering a therapeutically effective amount of a composition of theinvention, such as a VLPT antibody, polypeptide, and/or polynucleotide,to the individual.

In another embodiment, there is a method of inducing an immune responsein an individual, comprising the step of delivering to the individual atherapeutically effective amount of a VLPT polypeptide of the invention.In an additional embodiment of the present invention, there is a methodof inhibiting or preventing E. chaffeensis infection in a subjectcomprising the steps of: identifying a subject prior to exposure orsuspected of being exposed to or infected with E. chaffeensis; andadministering a polypeptide, antibody, and/or polynucleotide of theinvention in an amount effective to inhibit E. chaffeensis infection.

Polynucleotides of the invention may be comprised in a vector, such as aviral vector or a non-viral vector, wherein the viral vector may be anadenoviral vector, a retroviral vector, a lentiviral vector, anadeno-associated vector, a herpes virus vector, or a vaccinia virusvector and wherein the non-viral vector may be a plasmid. In furtheraspects of the invention, the vector comprise a promoter operably linkedto the polynucleotide wherein the promoter is operable in a prokaryote,a eukaryote, or both. The polynucleotide of the invention may becomprised in a liposome and/or comprised in a pharmaceuticallyacceptable excipient.

In certain aspects of the invention, there is an isolated antibody thatreacts immunologically to a polypeptide of the invention, and theantibody may be a monoclonal antibody, may be comprised in polyclonalantisera, or may be an antibody fragment, for example.

In other embodiments of the invention, there is a method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of a composition ofthe invention, such as a polypeptide, antibody and/or polynucleotide.

In additional embodiments of the invention, there is a method ofinhibiting E. chaffeensis infection in a subject comprising the stepsof: identifying a subject prior to exposure or suspected of beingexposed to or infected with E. chaffeensis; and administering thepolypeptide of the invention in an amount effective to inhibit E.chaffeensis infection. In further embodiments of the invention, there isa method of identifying an E. chaffeensis infection in an individual,comprising the step of assaying a sample from the individual for anantibody, polypeptide, and/or polynucleotide of the invention.

In specific aspects of the invention, a polypeptide is further definedas being from 10 to 11 amino acids in length, being from 10 to 12 aminoacids in length, being from 10 to 13 amino acids in length, being from10 to 14 amino acids in length, being from 10 to 15 amino acids inlength, being from 10 to 17 amino acids in length, from 10 to 20 aminoacids in length, from 10 to 25 amino acids in length, being from 14 to20 amino acids in length, being from 14 to 25 amino acids in length,being from 14 to 27 amino acids in length, being from 14 to 30 aminoacids in length, from 15 to 30 amino acids in length, being from 16 to20 amino acids in length, being from 16 to 25 amino acids in length,being from 16 to 30 amino acids in length, being from 17 to 20 aminoacids in length, being from 17 to 25 amino acids in length, being from17 to 30 amino acids in length, being from 20 to 25 amino acids inlength, being from 20 to 27 amino acids in length, being from 20 to 30amino acids in length, from 24 to 30 amino acids in length, from 24 to35 amino acids in length, from 24 to 40 amino acids in length, from 24to 45 amino acids in length, from 24 to 50 amino acids in length, from24 to 55 amino acids in length, from 24 to 60 amino acids in length,from 24 to 65 amino acids in length, from 24 to 70 amino acids inlength, from 24 to 75 amino acids in length, from 24 to 80 amino acidsin length, from 24 to 85 amino acids in length, from 24 to 90 aminoacids in length, from 24 to 95 amino acids in length, from 24 to 100amino acids in length, being from 30 to 50 amino acids in length, beingfrom 30 to 45 amino acids in length, or being from 30 to 55 amino acidsin length, for example.

In particular embodiments, a polypeptide of the invention is at least10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 or more amino acids inlength. In certain aspects of the invention, a polypeptide of theinvention is no more than 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or75 amino acids in length.

Variants of polypeptides comprising SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11, may bedefined as being at least 80% identical to SEQ ID NO:3; SEQ ID NO:4; SEQID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11; asbeing at least 85% identical to SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5;SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11; as being atleast 90% (or 91%, or 92%, or 93%, or 94%) identical to SEQ ID NO:3; SEQID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ IDNO:11; or as being at least 95% (or 96%, or 97%, or 98%, or 99%)identical to SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ IDNO:9; SEQ ID NO:10; or SEQ ID NO:11.

In an additional embodiment, there is a composition comprising: (a) apeptide having SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQID NO:9; SEQ ID NO:10; or SEQ ID NO:11; or (b) a variant of the peptideof (a), wherein the variant is at least 75% identical to SEQ ID NO:3;SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQID NO:11, wherein the composition is capable of eliciting an immunereaction in an individual. In a specific embodiment, there is a peptideis from 14 to 30 amino acids in length. In a specific embodiment, thereis a variant is further defined as being at least 80%, at least 85%, atleast 90%, or at least 95% identical to SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11.

A composition of the invention may be defined as having activity thatprovides immunity against Ehrlichia chaffeensis for an individual. Acomposition of the invention may be defined as having activity thatinduces an immune reaction against Ehrlichia chaffeensis for anindividual. Compositions of the invention include any polypeptide,peptide, polynucleotide, and/or antibody provided herein.

Nucleic acid molecules may be further defined as being comprised in avector, such as a viral vector or a non-viral vector, wherein the viralvector may comprise an adenoviral vector, a retroviral vector, or anadeno-associated viral vector. The nucleic acid molecule may becomprised in a liposome.

In specific embodiments, there is an isolated antibody thatimmunologically reacts with one or more of the amino acid sequencesselected from the group consisting of SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11. Infurther specific embodiments, the antibody is a monoclonal antibody, iscomprised in polyclonal antisera, or is an antibody fragment.

In an additional embodiment, there is a method of producing apolypeptide, comprising: providing a host cell comprising apolynucleotide of the invention and culturing the cell under conditionssuitable for the host cell to express the polynucleotide to produce theencoded polypeptide. The method may further comprise isolating thepolypeptide.

In an additional embodiment of the invention, there is a method ofinducing an immune response in an individual, comprising the step ofdelivering to the individual a therapeutically effective amount of acomposition of the invention.

In a further embodiment of the invention, there is a method ofinhibiting E. chaffeensis infection in a subject, comprising the step ofadministering to the subject prior to exposure or suspected of beingexposed to or infected with E. chaffeensis, an effective amount of acomposition of the invention.

In an additional embodiment of the invention, there is a method ofidentifying an E. chaffeensis infection in an individual, comprising thestep of assaying a sample from the individual for one or both of thefollowing: (a) a polypeptide of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5;SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11, or a mixturethereof; or (b) an antibody that immunologically reacts with an aminoacid sequence selected from the group consisting of SEQ ID NO:3; SEQ IDNO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ IDNO:11. In specific embodiments, the antibody of (b) immunologicallyreacts with an amino acid sequence of SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11. Inspecific aspects, assaying a sample for an antibody is further definedas assaying for an antibody by ELISA, such as by allowing assaying forone or more E. chaffeensis antibodies other then the antibody of (b).

In an embodiment of the invention, there is a kit, comprising one ormore of the following compositions: (a) an isolated polypeptidecomprising SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ IDNO:9; SEQ ID NO:10; or SEQ ID No: 11; (b) an isolated polypeptide thatis at least 70% identical to a polypeptide of (a); (c) an isolatedpolypeptide comprising SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ IDNO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11; (d) an isolatedpolypeptide that is at least 70% identical to SEQ ID NO:3; SEQ ID NO:4;SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11;(e) an isolated antibody that immunologically reacts with one or more ofthe amino acid sequences selected from the group consisting of SEQ IDNO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10;or SEQ ID NO:11. In a specific embodiment, the kit is further defined ascomprising two or more of the compositions.

In one embodiment of the present invention, there is a polypeptidecomposition, comprising one or more of the following: (a) an isolatedpolypeptide comprising one or more amino acid sequences selected fromthe group consisting of: SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ IDNO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11; or (b) an isolatedpolypeptide that is at least 95% identical to a polypeptide of (a). In aspecific embodiment, the isolated polypeptide is dispersed in apharmaceutically acceptable diluent. In another specific embodiment, thepolypeptide of (a) comprises the amino acid sequence selected from thegroup consisting of: SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In anadditional specific embodiment, the polypeptide of (a) comprises theamino acid sequence selected from the group consisting of: SEQ ID NO: 8;SEQ ID NO:9; SEQ ID NO:10; or SEQ ID NO:11.

In an additional embodiment of the invention, there is an isolatedantibody that immunologically reacts with one or more of the amino acidsequences selected from the group consisting of SEQ ID NO:3; SEQ IDNO:4; SEQ ID NO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ IDNO:11. In a specific embodiment, the antibody is a monoclonal antibodyor a polyclonal antibody.

In another embodiment of the invention, there is a method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of the composition ofthe invention. In one embodiment of the invention, there is a method ofinhibiting E. chaffeensis infection in a subject, comprising the step ofadministering to the subject an effective amount of a composition of theinvention.

In a certain aspect of the invention, there is a method of identifyingan E. chaffeensis infection in an individual, comprising the step ofassaying a sample from the individual for one of the following: (a) anisolated polypeptide comprising one or more amino acid sequencesselected from the group consisting of: SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:5; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11; or (b)an antibody that immunologically reacts with an amino acid sequenceselected from the group consisting of the polypeptides of (a). In oneembodiment of the invention, the sample is assayed for the polypeptideshaving the amino acid sequence of SEQ ID NO:17 and SEQ ID NO:19. In aspecific embodiment, the assay is by ELISA for the antibody of (b). Inanother embodiment of the invention, the method further comprisesobtaining the sample from the individual.

In an additional embodiment of the invention, there is a kit, housed ina suitable container, that comprises a polypeptide composition of theinvention. In some embodiments, the kit comprises two or morepolypeptide compositions of the invention. In a specific embodiment, thepolypeptide comprises the amino acid sequence of SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:3, SEQ ID NO:4, and/or SEQID NO:5.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features that are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying Figures. It is to be expressly understood, however, thateach of the Figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1A. provides an amino acid sequence of VLPT protein showing alldomains and location of four TRs (number of amino acids in parentheses;R=repeat) (including the N terminal 17 amino acids (SEQ ID NO:2),peptides R4 (SEQ ID NO:3), R3 (SEQ ID NO:4), R2 (SEQ ID NO:5) R1 (SEQ IDNO:6) and the C terminal 61 amino acids (SEQ ID NO:15). FIG. 1B.provides a Phylogenetic tree showing the relationship of the four E.chaffeensis VLPT repeats. The scale represents the amino acid percentidentity.

FIG. 2A shows the identification of native VLPT in E. chaffeensis wholecell lysates (lane 1), supernatants derived from E. chaffeensis infectedcells (lane 2), and E. canis whole cell lysates (lane 3) reacted withanti-VLPT-R3 peptide antibody and FIG. 2B shows the same, but withanti-E. chaffeensis dog serum. Pre-immunization rabbit serum or dogserum controls did not recognize E. chaffeensis whole cell lysates orsupernatants (data not shown).

FIG. 3 provides a schematic of synthetic and recombinant peptides usedto map the VLPT epitopes.

FIG. 4A shows the immunoreactivity of synthetic and recombinant peptidesof E. chaffeensis VLPT with anti-E. chaffeensis dog (no. 2251) serum.SDS-PAGE and total protein staining of purified recombinant peptides(top) and corresponding Western immunoblot probed with anti-E.chaffeensis dog serum (bottom). M, Precision Protein Standard (Bio-Rad);Ctrl, purified recombinant thioredoxin. FIG. 4B providesimmunoreactivity by ELISA of small recombinant and correspondingsynthetic VLPT polypeptides (N [synthetic only], R1, R2, R3, and R4) andlarge VLPT protein fragments (recombinant only; C, R4321-C, and R32).The OD readings represent the means for three wells (±standarddeviations), with the OD of the buffer-only wells subtracted.

FIG. 5A provides the sequence and orientation of overlapping peptides (7peptides) representing VLPT-R3 (SEQ ID NO:4), including peptides R3-1(SEQ ID NO:7), R3-2 (SEQ IN NO:8, R3-3 (SEQ ID NO:9), R3-4 (SEQ IDNO:10), R3-5 (SEQ ID NO:11), R3-6 (SEQ ID NO:12) and R3-7 (SEQ IDNO:13). FIG. 5B shows the immunoreactivity of VLPT-R3 overlappingpeptides by ELISA with anti-E. chaffeensis dog serum.

FIG. 6. Shows the immunoreactivity of synthetic and recombinant E.chaffeensis VLPT repeats (R2, R3 and R4) by ELISA with three HME patientsera (FIGS. 6A, 6B, 6C; Ctrl, purified recombinant thioredoxin).Synthetic E. chaffeensis VLPT-R3 reacted by ELISA with 14 HME patientsera (lanes 1-14), anti-E. chaffeensis dog serum (lane 15) and normalhuman serum (lane 16) (FIG. 6D). The normal human serum did notrecognize other peptides and proteins as well (data not shown).

FIG. 7. Western immunoblot of DH82 cell culture supernatant (0 to 6 dayspostinfection, lanes 1 to 7, respectively) of E. chaffeensis probed withanti-VLPT-R3 peptide antibody. M, Precision Protein Standard (Bio-Rad).

FIG. 8A provides an electron photomicrograph of an ultrathin section ofE. chaffeensis-infected DH82 cells demonstrating E. chaffeensis VLPTlocalization in reticulate and dense-cored ehrlichiae, and FIG. 8Bprovides a corresponding ultrathin section containing uninfected DH82cells (negative control). Cells in both panels were reacted with rabbitanti-VLPT-R3 peptide antibody (1:10,000). Bar=1 μm

DETAILED DESCRIPTION OF THE INVENTION

The present application incorporates by reference in their entiretyPCT/US2007/75343, filed Aug. 7, 2007, and U.S. Provisional PatentApplication Ser. No. 60/841,465, filed Aug. 31, 2006.

I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “carbohydrate” as used herein refers to a composition comprisedof carbon, hydrogen, and oxygen, particularly in the ratio of 2H:1C:10.The term includes sugars, starches, and celluloses, for example.

The term “epitope” as used herein refers to a site of a composition towhich a specific antibody binds.

The term “glycan,” which may also be referred to as a “polysaccharide,”as used herein refers to a carbohydrate that can be decomposed byhydrolysis into two or more monosaccharides. In other words, it may bereferred to as a chain of simple sugars (aldehyde or ketone derivativesof a polyhydric alcohol).

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or between polypeptides, as the case maybe, as determined by the number of matches between strings of two ormore nucleotide residues or two or more amino acid residues. “Identity”measures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”).

The term “immunogenic” as used herein refers to a composition that isable to provoke an immune response against it.

The term “immune response” as used herein refers to the reaction of theimmune system to the presence of an antigen by making antibodies to theantigen. In further specific embodiments, immunity to the antigen may bedeveloped on a cellular level, by the body as a whole, hypersensitivityto the antigen may be developed, and/or tolerance may be developed, suchas from subsequent challenge. In specific embodiments, an immuneresponse entails lymphocytes identifying an antigenic molecule asforeign and inducing the formation of antibodies and lymphocytes capableof reacting with it and rendering it less harmful.

The term “immunoreactive” as used herein refers to a composition beingreactive with antibodies from the sera of an individual. In specificembodiments, a composition is immunoreactive if an antibody recognizesit, such as by binding to it and/or immunologically reacting with it.

The term “mucin” as used herein refers to one or more highlyglycosylated glycoproteins with N-acetylgalactosamine (GalNAc.)

The term “ortholog” as used herein refers to a polynucleotide from onespecies that corresponds to a polynucleotide in another species; the twopolynucleotides are related through a common ancestral species (ahomologous polynucleotide). However, the polynucleotide from one specieshas evolved to become different from the polynucleotide of the otherspecies.

The term “similarity” is a related concept, but in contrast to“identity”, refers to a sequence relationship that includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If, in the sameexample, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% ( 15/20). Therefore, in cases where there areconservative substitutions, the degree of similarity between twopolypeptides will be higher than the percent identity between those twopolypeptides.

The term “subunit vaccine” as used herein refers to a vaccine wherein apolypeptide or fragment thereof is employed, as opposed to an entireorganism.

The term “vaccine” as used herein refers to a composition that providesimmunity to an individual upon challenge.

The term “virulence factor” as used herein refers to one or more geneproducts that enable a microorganism to establish itself on or within aparticular host species and enhance its pathogenicity. Exemplaryvirulence factors include, for example, cell surface proteins thatmediate bacterial attachment, cell surface carbohydrates and proteinsthat protect a bacterium, bacterial toxins, and hydrolytic enzymes thatmay contribute to the pathogenicity of the bacterium.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and so forth which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984),ANIMAL CELL CULTURE (R. I. Freshney, Ed., 1987), the series METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. M. Miller and M. P. Calos eds. 1987), HANDBOOK OF EXPERIMENTALIMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.), CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,J. G. Siedman, J. A. Smith, and K. Struhl, eds., 1987), CURRENTPROTOCOLS IN IMMUNOLOGY (J. E. coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, eds., 1991); ANNUAL REVIEW OFIMMUNOLOGY; as well as monographs in journals such as ADVANCES INIMMUNOLOGY. All patents, patent applications, and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference.

II. Embodiments of the Present Invention

The present invention concerns compositions and methods related toEhrlichia spp. Proteins, the polynucleotides that encode them, andfragments and related molecules thereto. In particular aspects of theinvention, there are differentially-expressed and secreted majorimmunoreactive protein orthologs of E. canis and E. chaffeensis thatelicit early antibody responses to epitopes on glycosylated tandemrepeats. Specifically, the present invention concerns one or moreglycoproteins from Ehrlichia spp., in specific embodiments. In furtherembodiments, the present invention relates to a glycoprotein fromEhrlichia spp. that is a VLPT protein. In additional embodiments, theVLPT protein is from E. chaffeensis.

Ehrlichia chaffeensis has a small subset of major immunoreactiveproteins that includes a 19-kDa protein that elicits an early ehrlichialspecific antibody response in infected dogs. The present inventionconcerns the identification and molecular characterization of the E.chaffeensis variable-length PCR target (VLPT) protein.

Some embodiments of the present invention are directed toward a methodof inhibiting E. chaffeensis infection in a subject comprising the stepsof identifying a subject prior to exposure or suspected of being exposedto or infected with E. chaffeensis and administering a compositioncomprising an antigen of E. chaffeensis in an amount effective toinhibit E. chaffeensis infection. The inhibition may occur through anymeans such as e.g., the stimulation of the subject's humoral or cellularimmune responses, or by other means such as inhibiting the normalfunction of the antigen, or even competing with the antigen forinteraction with some agent in the subject's body, or a combinationthereof, for example.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyand a therapeutic moiety, and wherein the targeting moiety is specificfor VLPT protein. In certain aspects, the targeting moiety is anantibody specific for VLPT or ligand or ligand binding domain that bindsVLPT Likewise, the therapeutic moiety may comprise a radioisotope, atoxin, a chemotherapeutic agent, an immune stimulant, a cytotoxic agent,or an antibiotic, for example.

Other embodiments of the present invention concern diagnosis ofehrlichial infection in a mammal by assaying a sample from the mammal,such as blood or serum, for example, for antibodies to a VLPTcomposition (for E. chaffeensis).

III. E. chaffeensis VLPT Amino Acid Compositions

The present invention regards a polypeptide or peptide comprising E.chaffeensis VLPT. For the sake of brevity, the following section willrefer to any E. chaffeensis VLPT amino acid compositions of the presentinvention, including polypeptides and peptides.

In particular embodiments, a polypeptide may be a recombinantpolypeptide or it may be isolated and/or purified from nature, forexample. In particular aspects, the amino acid sequence is encoded by anucleic acid sequence. The polypeptide is useful as an antigen, inspecific embodiments. In other particular embodiments, a peptide may begenerated synthetically or encoded by an oligonucleotide, for example.The peptide is useful as an antigen, in specific embodiments.

The present invention is also directed towards a method of producing therecombinant polypeptide, comprising the steps of obtaining a vector thatcomprises an expression construct comprising a sequence encoding theamino acid sequence operatively linked to a promoter; transfecting thevector into a cell; and culturing the cell under conditions effectivefor expression of the expression construct. The amino acid sequence maybe generated synthetically, in alternative embodiments.

By a “substantially pure protein” is meant a protein that has beenseparated from at least some of those components that naturallyaccompany it. A substantially pure immunoreactive composition may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding an immunoreactivecomposition; or by chemically synthesizing the protein, for example.Accordingly, substantially pure proteins include proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

Thus, in certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 130 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, or greater amino acid residues, and any range derivabletherein.

As used herein, an “amino acid molecule” refers to any polypeptide,polypeptide derivative, or polypeptide mimetic as would be known to oneof ordinary skill in the art. In certain embodiments, the residues ofthe proteinaceous molecule are sequential, without any non-amino acidmolecule interrupting the sequence of amino acid molecule residues. Inother embodiments, the sequence may comprise one or more non-aminomolecule moieties. In particular embodiments, the sequence of residuesof the proteinaceous molecule may be interrupted by one or morenon-amino molecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1, below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3- Aminoadipicacid Hyl Hydroxylysine Bala β-alanine, β-Amino- AHyl allo-Hydroxylysinepropionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments, theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance that produces no significant untoward effects when applied to,or administered to, a given organism according to the methods andamounts described herein. Such untoward or undesirable effects are thosesuch as significant toxicity or adverse immunological reactions.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials, for example. Thenucleotide and protein, polypeptide and peptide sequences for variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. Two suchdatabases are the National Center for Biotechnology Information'sGenBank® and GenPept databases, for example. The coding regions forthese known genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides and peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or protein, polypeptide,or peptide composition that has been subjected to fractionation toremove various other proteins, polypeptides, or peptides, and whichcomposition substantially retains its activity, as may be assessed, forexample, by the protein assays, as would be known to one of ordinaryskill in the art for the specific or desired protein, polypeptide orpeptide. Exemplary activities that may be assessed for retention in thepurified proteinaceous composition are iron-binding activity andimmunoreactivity.

In specific embodiments of the present invention, a polypeptide islabeled, and any detectable label is suitable in the invention. Thelabel may be attached to the polypeptide at the N-terminus, at theC-terminus, or in a side chain of an amino acid residue, for example.One or more labels may be employed. Exemplary labels includedradioactive labels, fluorescent labels, colorimetric labels, and soforth. In specific embodiments, the label is covalently attached to thepolypeptide.

IV. E. chaffeensis VLPT Nucleic Acid Compositions

Certain embodiments of the present invention concern an E. chaffeensisVLPT nucleic acid. For the sake of brevity, the following section willrefer to any E. chaffeensis VLPT nucleic acid compositions of thepresent invention.

In certain aspects, a nucleic acid comprises a wild-type or a mutantnucleic acid. In particular aspects, a nucleic acid encodes for orcomprises a transcribed nucleic acid. In other aspects, a nucleic acidcomprises a nucleic acid segment, or a biologically functionalequivalent thereof. In particular aspects, a nucleic acid encodes aprotein, polypeptide, peptide.

The term “nucleic acid” is well known in the art and may be usedinterchangeably herein with the term “polynucleotide.” A “nucleic acid”as used herein will generally refer to a molecule (i.e., a strand) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

A. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, carboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art.

B. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

C. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

D. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947 which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167 which describe nucleic acids incorporating fluorescent analogsof nucleosides found in DNA or RNA, particularly for use as fluorescentnucleic acids probes; U.S. Pat. No. 5,614,617 which describesoligonucleotide analogs with substitutions on pyrimidine rings thatpossess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232and 5,859,221 which describe oligonucleotide analogs with modified5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties) used innucleic acid detection; U.S. Pat. No. 5,446,137 which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165 whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606 which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697 which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847 which describe the linkage of a substituentmoeity which may comprise a drug or label to the 2′ carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties; U.S. Pat. No. 5,223,618 whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967 which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240 which describe oligonucleotides with three or four atom linkermoeity replacing phosphodiester backbone moeity used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988 which describes hydrophobic carrier agent attached tothe 2′-O position of oligonucleotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136 which describesoligonucleotides conjugated to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154 which describes RNA linkedto a DNA to form a DNA-RNA hybrid.

E. Polyether and Peptide Nucleic Acids

In certain embodiments, it is contemplated that a nucleic acidcomprising a derivative or analog of a nucleoside or nucleotide may beused in the methods and compositions of the invention. A non-limitingexample is a “polyether nucleic acid”, described in U.S. Pat. No.5,908,845, incorporated herein by reference. In a polyether nucleicacid, one or more nucleobases are linked to chiral carbon atoms in apolyether backbone.

Another non-limiting example is a “peptide nucleic acid”, also known asa “PNA”, “peptide-based nucleic acid analog” or “PENAM”, described inU.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336,5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which isincorporated herein by reference. Peptide nucleic acids generally haveenhanced sequence specificity, binding properties, and resistance toenzymatic degradation in comparison to molecules such as DNA and RNA(Egholm et al., 1993; PCT/EP/01219). A peptide nucleic acid generallycomprises one or more nucleotides or nucleosides that comprise anucleobase moiety, a nucleobase linker moeity that is not a 5-carbonsugar, and/or a backbone moiety that is not a phosphate backbone moiety.Examples of nucleobase linker moieties described for PNAs include azanitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat.No. 5,539,082). Examples of backbone moieties described for PNAs includean aminoethylglycine, polyamide, polyethyl, polythioamide,polysulfinamide or polysulfonamide backbone moiety.

In certain embodiments, a nucleic acid analogue such as a peptidenucleic acid may be used to inhibit nucleic acid amplification, such asin PCR, to reduce false positives and discriminate between single basemutants, as described in U.S. Pat. No. 5,891,625. Other modificationsand uses of nucleic acid analogs are known in the art, and areencompassed by the VLPT polynucleotide. In a non-limiting example, U.S.Pat. No. 5,786,461 describes PNAs with amino acid side chains attachedto the PNA backbone to enhance solubility of the molecule. In anotherexample, the cellular uptake property of PNAs is increased by attachmentof a lipophilic group. U.S. application Ser. No. 117,363 describesseveral alkylamino moeities used to enhance cellular uptake of a PNA.Another example is described in U.S. Pat. Nos. 5,766,855, 5,719,262,5,714,331 and 5,736,336, which describe PNAs comprising naturally andnon-naturally occurring nucleobases and alkylamine side chains thatprovide improvements in sequence specificity, solubility and/or bindingaffinity relative to a naturally occurring nucleic acid.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemically synthesis usingphosphotriester, phosphite or phosphoramidite chemistry and solid phasetechniques such as described in EP 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 1989,incorporated herein by reference).

G. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al., 1989, incorporatedherein by reference).

In certain aspect, the present invention concerns a nucleic acid that isan isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

H. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the peptide or polypeptide sequence. Thus, a “nucleic acidsegment” may comprise any part of a gene sequence, of from about 2nucleotides to the full length of the peptide or polypeptide encodingregion.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe generated:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10 mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and soon. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15,2 to 16, 3 to 17 . . . and so on. For a 20-mer, the nucleic segmentscorrespond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. Incertain embodiments, the nucleic acid segment may be a probe or primer.As used herein, a “probe” generally refers to a nucleic acid used in adetection method or composition. As used herein, a “primer” generallyrefers to a nucleic acid used in an extension or amplification method orcomposition.

I. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to one or more other nucleic acids. In specificembodiments, for example, a nucleic acid is employed for antisense orsiRNA purposes, such as to inhibit at least partially expression of apolynucleotide.

In particular embodiments the invention encompasses a nucleic acid or anucleic acid segment complementary to the sequence set forth herein, forexample. A nucleic acid is “complement(s)” or is “complementary” toanother nucleic acid when it is capable of base-pairing with anothernucleic acid according to the standard Watson-Crick, Hoogsteen orreverse Hoogsteen binding complementarity rules. As used herein “anothernucleic acid” may refer to a separate molecule or a spatial separatedsequence of the same molecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

J. Hybridization

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl, forexample, at temperatures of about 50° C. to about 70° C. or, forexample, wherein said stringent conditions are hybridization at 50-65°C., 5×SSPC, 50% formamide; wash 50-65° C., 5×SSPC; or wash at 60° C.,0.5×SSC, 0.1% SDS. It is understood that the temperature and ionicstrength of a desired stringency are determined in part by the length ofthe particular nucleic acid(s), the length and nucleobase content of thetarget sequence(s), the charge composition of the nucleic acid(s), andto the presence or concentration of formamide, tetramethylammoniumchloride or other solvent(s) in a hybridization mixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

V. Nucleic Acid-Based Expression Systems

In particular embodiments, the present invention concerns apolynucleotide that encodes an immunoreactive ehrlichiae polypeptide,and also includes delivering the polynucleotide encoding thepolypeptide, or encoded product thereof, to an individual in needthereof, such as an individual infected with Erhlichia and/or anindividual susceptible to being infected with Erhlichia. For the sake ofbrevity, the following section will refer to any E. chaffeensis VLPTnucleic acid compositions and/or nucleic acid-based expression system ofthe present invention.

The present invention is directed toward substantially pure and/orisolated DNA sequence encoding an immunoreactive Ehrlichia composition.Generally, the encoded protein comprises an N-terminal sequence, whichmay be cleaved after post-translational modification resulting in theproduction of mature protein.

It is well-known in the art that because of the degeneracy of thegenetic code (i.e., for most amino acids, more than one nucleotidetriplet (codon) codes for a single amino acid), different nucleotidesequences can code for a particular amino acid, or polypeptide. Thus,the polynucleotide sequences of the subject invention include any of theprovided exemplary sequences or a degenerate variant of such a sequence,for example. In particular aspects of the invention, a degeneratevariant comprises a sequence that is not identical to a sequence of theinvention but that still retains one or more properties of a sequence ofthe invention.

As used herein, “substantially pure DNA” means DNA that is not part of amilieu in which the DNA naturally occurs, by virtue of separation(partial or total purification) of some or all of the molecules of thatmilieu, or by virtue of alteration of sequences that flank the claimedDNA. The term therefore includes, for example, a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote; or thatexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA, which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein.

The present invention is further directed to an expression vectorcomprising a polynucleotide encoding an immunoreactive Ehrlichiacomposition and capable of expressing the polynucleotide when the vectoris introduced into a cell. In specific embodiments, the vector comprisesin operable linkage the following: a) an origin of replication; b) apromoter; and c) a DNA sequence coding for the protein.

As used herein “vector” may be defined as a replicable nucleic acidconstruct, e.g., a plasmid or viral nucleic acid. Vectors may be used toamplify and/or express nucleic acid encoding an immunoreactivecomposition of Ehrlichia. An expression vector is a replicable constructin which a nucleic acid sequence encoding a polypeptide is operablylinked to suitable control sequences capable of effecting expression ofthe polypeptide in a cell. The need for such control sequences will varydepending upon the cell selected and the transformation method chosen.Generally, control sequences include a transcriptional promoter and/orenhancer, suitable mRNA ribosomal binding sites, and sequences thatcontrol the termination of transcription and translation, for example.Methods that are well-known to those skilled in the art can be used toconstruct expression vectors comprising appropriate transcriptional andtranslational control signals. See for example, the techniques describedin Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2ndEd.), Cold Spring Harbor Press, N.Y. A polynucleotide sequence to beexpressed and its transcription control sequences are defined as being“operably linked” if the transcription control sequences effectivelycontrol the transcription of the polynucleotide sequence. Vectors of theinvention include, but are not limited to, plasmid vectors and viralvectors. Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses, for example.

In general, expression vectors comprise promoter sequences thatfacilitate the efficient transcription of the polynucleotide to beexpressed, are used in connection with a host cell. As used herein, theterm “host” is meant to include not only prokaryotes but alsoeukaryotes, such as yeast, plant and animal cells. A recombinantpolynucleotide that encodes an immunoreactive composition of Ehrlichiaof the present invention can be used to transform a host using any ofthe techniques commonly known to those of ordinary skill in the art.Prokaryotic hosts may include E. coli, S. tymphimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts, suchas Pichia pastoris, mammalian cells and insect cells.

The following description concerns exemplary elements, reagents, andmethods for polynucleotides and nucleic acid delivery of an Ehrlichiapolynucleotide.

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the betalactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the cell,organelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

The promoter may be one suitable for use in a prokaryotic cell, aeukaryotic cell, or both. Additionally any promoter/enhancer combination(as per, for example, the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is one possible embodiment.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with betagalactosidase, ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

10. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Components of the present invention may comprise aviral vector that encode one or more compositions or other componentssuch as, for example, an immunomodulator or adjuvant. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

a. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

b. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the compositions of thepresent invention as it has a high frequency of integration and it caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue culture (Muzyczka, 1992) orin vivo. AAV has a broad host range for infectivity (Tratschin et al.,1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,1988). Details concerning the generation and use of rAAV vectors aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference.

c. Retroviral Vectors

Retroviruses have useful as delivery vectors due to their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding a composition of interest) is inserted into the viral genome inthe place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

d. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

e. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

11. Vector Delivery and Cell Transformation

Suitable methods for ehrlichial nucleic acid delivery for transformationof an organelle, a cell, a tissue or an organism for use with thecurrent invention are believed to include virtually any method by whicha nucleic acid (e.g., DNA) can be introduced into an organelle, a cell,a tissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.,1986; Potter et al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

a. Ex vivo Transformation

Methods for tranfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example, canine endothelial cells have been genetically altered byretrovial gene transfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, yucatan minipig endothelial cellswere transfected by retrovirus in vitro and transplanted into an arteryusing a double-ballonw catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and transfected exvivo using the nucleic acids of the present invention. In particularaspects, the transplanted cells or tissues may be placed into anorganism. In preferred facets, a nucleic acid is expressed in thetransplanted cells or tissues.

b. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection), such as, for example, subcutaneously, intradermally,intramuscularly, intervenously, intraperitoneally, etc. Methods ofinjection of vaccines are well known to those of ordinary skill in theart (e.g., injection of a composition comprising a saline solution).Further embodiments of the present invention include the introduction ofa nucleic acid by direct microinjection. Direct microinjection has beenused to introduce nucleic acid constructs into Xenopus oocytes (Harlandand Weintraub, 1985). The amount of composition used may vary upon thenature of the antigen as well as the organelle, cell, tissue or organismused

c. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high voltage electric discharge. In some variantsof this method, certain cell wall degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cellsmore susceptible to transformation by electroporation than untreatedcells (U.S. Pat. No. 5,384,253, incorporated herein by reference).Alternatively, recipient cells can be made more susceptible totransformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre B lymphocytes have been transfected with humankappa immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

d. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV 1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

e. DEAE Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

f. Sonication Loading

Additional embodiments of the present invention include the introductionof a nucleic acid by direct sonic loading. LTK fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

g. Liposome-Mediated Transfection

In a further embodiment of the invention, an ehrlichial nucleic acid maybe comprised with a lipid complex such as, for example, comprised in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL)or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry of liposomeencapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposomemay be complexed or employed in conjunction with nuclear non histonechromosomal proteins (HMG 1) (Kato et al., 1991). In yet furtherembodiments, a liposome may be complexed or employed in conjunction withboth HVJ and HMG 1. In other embodiments, a delivery vehicle maycomprise a ligand and a liposome.

h. Receptor-Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor mediated gene targeting vehicles comprise a cellreceptor specific ligand and a nucleic acid binding agent. Otherscomprise a cell receptor specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell specific binding. For example, lactosyl ceramide, a galactoseterminal asialganglioside, have been incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes(Nicolau et al., 1987). It is contemplated that the tissue specifictransforming constructs of the present invention can be specificallydelivered into a target cell in a similar manner.

i. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

Microprojectile bombardment may be used to transform various cell(s),tissue(s) or organism(s), such as for example any plant species.Examples of species which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinelli, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into a cell(e.g., a plant cell) by acceleration is the Biolistics Particle DeliverySystem, which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with cells, such as for example, a monocot plantcells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

12. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with acomposition of the invention. The tissue may be part or separated froman organism. In certain embodiments, a tissue may comprise, but is notlimited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, liver, lung, lymph node, muscle,neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, smallintestine, spleen, stem cells, stomach, testes, anthers, ascite tissue,cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen,root tips, roots, silk, stalks, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art(see, for example, webpage world wide web atphylogeny.arizona.edu/tree/phylogeny.html).

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials (world wide web at atcc.org). Anappropriate host can be determined by one of skill in the art based onthe vector backbone and the desired result. A plasmid or cosmid, forexample, can be introduced into a prokaryote host cell for replicationof many vectors. Cell types available for vector replication and/orexpression include, but are not limited to, bacteria, such as E. coli(e.g., E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776(ATCC No. 31537) as well as E. coli W3110 (F, lambda, prototrophic, ATCCNo. 273325), DH5.alpha., JM109, and KCB, bacilli such as Bacillussubtilis; and other enterobacteriaceae such as Salmonella typhimurium,Serratia marcescens, various Pseudomonas specie, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK Gold Cells (STRATAGENE®, La Jolla). In certain embodiments,bacterial cells such as E. coli LE392 are particularly contemplated ashost cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

13. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methodsare preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

In some embodiments, the expressed proteinaceous sequence forms aninclusion body in the host cell, the host cells are lysed, for example,by disruption in a cell homogenizer, washed and/or centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed. Inclusion bodies may be solubilizedin solutions containing high concentrations of urea (e.g. 8M) orchaotropic agents such as guanidine hydrochloride in the presence ofreducing agents, such as beta mercaptoethanol or DTT (dithiothreitol),and refolded into a more desirable conformation, as would be known toone of ordinary skill in the art.

VI. Biological Functional Equivalents

As modifications and/or changes may be made in the structure of thepolynucleotides and/or proteins according to the present invention,while obtaining molecules having similar or improved characteristics,such biologically functional equivalents are also encompassed within thepresent invention.

A. Modified Polynucleotides and Polypeptides

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard protein.This can be accomplished to the degeneracy of the genetic code, i.e.,the presence of multiple codons, which encode for the same amino acids.In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide made be (and encode) a biologicalfunctional equivalent with more significant changes. Certain amino acidsmay be substituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies, binding sites onsubstrate molecules, receptors, and such like. So-called “conservative”changes do not disrupt the biological activity of the protein, as thestructural change is not one that impinges of the protein's ability tocarry out its designed function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity. Biologically functionalequivalents are thus defined herein as those proteins (andpolynucleotides) in selected amino acids (or codons) may be substituted.Functional activity.

In general, the shorter the length of the molecule, the fewer changesthat can be made within the molecule while retaining function. Longerdomains may have an intermediate number of changes. The full-lengthprotein will have the most tolerance for a larger number of changes.However, it must be appreciated that certain molecules or domains thatare highly dependent upon their structure may tolerate little or nomodification.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are alla similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8);tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2);glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5);lysine (3.9); and/or arginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (0.4);proline (−0.5±1); alanine (0.5); histidine (0.5); cysteine (1.0);methionine (1.3); valine (1.5); leucine (1.8); isoleucine (1.8);tyrosine (2.3); phenylalanine (2.5); tryptophan (3.4). In making changesbased upon similar hydrophilicity values, the substitution of aminoacids whose hydrophilicity values are within ±2 is preferred, thosewhich are within ±1 are particularly preferred, and/or those within ±0.5are even more particularly preferred.

B. Altered Amino Acids

The present invention, in many aspects, relies on the synthesis ofpeptides and polypeptides in cyto, via transcription and translation ofappropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof. However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. Table 1 provides exemplary,but not limiting, modified and/or unusual amino acids

C. Mimetics

In addition to the biological functional equivalents discussed above,the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins. Vita et al. (1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. Weisshoff et al. (1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

VII. Immunological Compositions

In particular embodiments of the invention, immunological compositionsare employed. For the sake of brevity, the following section will referto any E. chaffeensis VLPT immunological compositions of the presentinvention, such as are described elsewhere herein as only exemplaryembodiments. For example, the compositions may include all or part of anE. chaffeensis VLPT polypeptide, such as one comprising part or all ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, a VLPTpolynucleotide, such as one comprising part or all of SEQ ID NO:16, anantibody to a polypeptide or peptide of the invention, or a mixturethereof, for example. Antibodies may be utilized to bind an antigen,thereby rendering the molecule at least partially ineffective for itsactivity, for example. In other embodiments, antibodies to the antigenare employed in diagnostic aspects of the invention, such as fordetecting the presence of the antigen from a sample. Exemplary samplesmay be from an animal suspected of having E. canis or E. chaffeensisinfection, from an animal susceptible to E. canis or E. chaffeensisinfection, or from an animal that has an E. canis or E. chaffeensisinfection. Exemplary samples may be obtained from blood, serum,cerebrospinal fluid, urine, feces, cheek scrapings, nipple aspirate, andso forth.

Purified immunoreactive compositions or antigenic fragments of theimmunoreactive compositions can be used to generate new antibodies or totest existing antibodies (e.g., as positive controls in a diagnosticassay) by employing standard protocols known to those skilled in theart.

As is well known in the art, immunogenicity to a particular immunogencan be enhanced by the use of non-specific stimulators of the immuneresponse known as adjuvants. Exemplary and preferred adjuvants includecomplete BCG, Detox, (RIBI, Immunochem Research Inc.), ISCOMS andaluminum hydroxide adjuvant (Superphos, Biosector).

Included in this invention are polyclonal antisera generated by usingthe immunoreactive composition or a fragment of the immunoreactivecomposition as an immunogen in, e.g., rabbits. Standard protocols formonoclonal and polyclonal antibody production known to those skilled inthis art are employed. The monoclonal antibodies generated by thisprocedure can be screened for the ability to identify recombinantEhrlichia cDNA clones, and to distinguish them from known cDNA clones,for example.

The invention encompasses not only an intact monoclonal antibody, butalso an immunologically-active antibody fragment, e.g., a Fab or (Fab)2fragment; an engineered single chain scFv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

In one embodiment, the antibody, or fragment thereof, may be linked to atoxin or to a detectable label, e.g. a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label or colorimetric label. Examplesof suitable toxins include diphtheria toxin, Pseudomonas exotoxin A,ricin, and cholera toxin. Examples of suitable enzyme labels includemalate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,alcohol dehydrogenase, alpha glycerol phosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase,etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986)Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16, 93-95; Wesby et al., (1984) Physiol. Chem. Phys. Med. NMR16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examplesof suitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an opthaldehyde label, afluorescamine label, etc. Examples of chemiluminiscent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,a luciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of these and other suitablelabels, which may be employed in accordance with the present invention.The binding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedyet al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al., (1977)Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the laterare the glutaraldehyde method, the periodate method, the dimaleimidemethod, the maleimidobenzyl-N-hydroxy-succinimde ester method. All ofthese methods are incorporated by reference herein.

D. Antibodies

In certain aspects of the invention, one or more antibodies may beproduced to the expressed VLPT. These antibodies may be used in variousdiagnostic and/or therapeutic applications described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent invention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126.

Antibody-like binding peptidomimetics are also contemplated in thepresent invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003March; 49(2):209-16 describe “antibody like binding peptidomimetics”(ABiPs), which are peptides that act as pared-down antibodies and havecertain advantages of longer serum half-life as well as less cumbersomesynthesis methods.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin.

E. Exemplary Methods for Generating Monoclonal Antibodies

Exemplary methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal witha LEE or CEE composition in accordance with the present invention andcollecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,.gamma.-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, suchas thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipidA (MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/TWEEN 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as γ-interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60 61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3×63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U, MPC11, MPC11×45 GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3,Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICR LON HMy2 andUC729 6 are all useful in connection with human cell fusions. See Yoo etal., J Immunol Methods. 2002 Mar. 1; 261(1-2):1-20, for a discussion ofmyeloma expression systems.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the 8azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. In one embodiment, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies. In another example, LEEs orCEEs can be used to produce antigens in vitro with a cell free system.These can be used as targets for scanning single chain antibodylibraries. This would enable many different antibodies to be identifiedvery quickly without the use of animals.

Another embodiment of the invention for producing antibodies accordingto the present invention is found in U.S. Pat. No. 6,091,001, whichdescribes methods to produce a cell expressing an antibody from agenomic sequence of the cell comprising a modified immunoglobulin locususing Cre-mediated site-specific recombination is disclosed. The methodinvolves first transfecting an antibody-producing cell with ahomology-targeting vector comprising a lox site and a targeting sequencehomologous to a first DNA sequence adjacent to the region of theimmunoglobulin loci of the genomic sequence which is to be converted toa modified region, so the first lox site is inserted into the genomicsequence via site-specific homologous recombination. Then the cell istransfected with a lox-targeting vector comprising a second lox sitesuitable for Cre-mediated recombination with the integrated lox site anda modifying sequence to convert the region of the immunoglobulin loci tothe modified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

F. Antibody Conjugates

The present invention further provides antibodies against VLPT proteins,polypeptides and peptides, generally of the monoclonal type, that arelinked to at least one agent to form an antibody conjugate. In order toincrease the efficacy of antibody molecules as diagnostic or therapeuticagents, it is conventional to link or covalently bind or complex atleast one desired molecule or moiety. Such a molecule or moiety may be,but is not limited to, at least one effector or reporter molecule.Effector molecules comprise molecules having a desired activity, e.g.,cytotoxic activity. Non-limiting examples of effector molecules whichhave been attached to antibodies include toxins, anti-tumor agents,therapeutic enzymes, radio-labeled nucleotides, antiviral agents,chelating agents, cytokines, growth factors, and oligo- orpoly-nucleotides. By contrast, a reporter molecule is defined as anymoiety which may be detected using an assay. Non-limiting examples ofreporter molecules which have been conjugated to antibodies includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, colored particles or ligands, suchas biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic or anticellular agent, and may be termed “immunotoxins”.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴-carbon, ⁵¹chromium,³⁶-chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium186, rhenium188, ⁷⁵selenium, ³⁵sulphur, technicium99m and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled with technetium byligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa ALEXA 350, Alexa ALEXA 430, AMCA, BODIPY 630/650, BODIPY 650/665,BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3,Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488,Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green,Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine,and/or Texas Red

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6 α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In another embodiment of the invention, the anti-VLPT antibodies arelinked to semiconductor nanocrystals such as those described in U.S.Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all ofwhich are incorporated herein in their entireties); as well as PCTPublication No. 99/26299 (published May 27, 1999). In particular,exemplary materials for use as semiconductor nanocrystals in thebiological and chemical assays of the present invention include, but arenot limited to those described above, including group II-VI, III-V andgroup IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si andternary and quaternary mixtures thereof. Methods for linkingsemiconductor nanocrystals to antibodies are described in U.S. Pat. Nos.6,630,307 and 6,274,323.

G. Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asimmunoreactive polypeptides. The antibodies prepared in accordance withthe present invention may be employed to detect wild type and/or mutantproteins, polypeptides and/or peptides. The use of wild-type and/ormutant antibodies is contemplated. Some immunodetection methods includeenzyme linked immunosorbent assay (ELISA), radioimmunoas say (RIA),immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay,bioluminescent assay, and Western blot to mention a few. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamuraet al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of comprising protein, polypeptide and/or peptide, andcontacting the sample with a first anti-VLPT antibody in accordance withthe present invention, as the case may be, under conditions effective toallow the formation of immunocomplexes.

These methods include methods for purifying wild type and/or mutantproteins, polypeptides and/or peptides as may be employed in purifyingwild type and/or mutant proteins, polypeptides and/or peptides frompatients' samples and/or for purifying recombinantly expressed wild typeor mutant proteins, polypeptides and/or peptides. In these instances,the antibody removes the antigenic wild type and/or mutant protein,polypeptide and/or peptide component from a sample. The antibody willpreferably be linked to a solid support, such as in the form of a columnmatrix, and the sample suspected of containing the wild type or mutantprotein antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody, which wild type ormutant protein antigen is then collected by removing the wild type ormutant protein and/or peptide from the column.

The immunobinding methods also include methods for detecting andquantifying the amount of a wild type or mutant protein reactivecomponent in a sample and the detection and quantification of any immunecomplexes formed during the binding process. Here, one would obtain asample suspected of comprising a wild type or mutant protein and/orpeptide or suspected of comprising an E. canis organism, and contact thesample with an antibody against wild type or mutant, and then detect andquantify the amount of immune complexes formed under the specificconditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing a wild type or mutantprotein-specific antigen, such as a specimen, a homogenized tissueextract, a cell, separated and/or purified forms of any of the abovewild type or mutant protein-containing compositions, or even anybiological fluid that comes into contact with an E. canis organism uponinfection.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any proteinantigens present. After this time, the sample-antibody composition, suchas a tissue section, ELISA plate, dot blot or western blot, willgenerally be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firststep biotinylated, monoclonal or polyclonal antibody is used to detectthe target antigen(s), and a second step antibody is then used to detectthe biotin attached to the complexed biotin. In that method the sampleto be tested is first incubated in a solution containing the first stepantibody. If the target antigen is present, some of the antibody bindsto the antigen to form a biotinylated antibody/antigen complex. Theantibody/antigen complex is then amplified by incubation in successivesolutions of streptavidin (or avidin), biotinylated DNA, and/orcomplementary biotinylated DNA, with each step adding additional biotinsites to the antibody/antigen complex. The amplification steps arerepeated until a suitable level of amplification is achieved, at whichpoint the sample is incubated in a solution containing the second stepantibody against biotin. This second step antibody is labeled, as forexample with an enzyme that can be used to detect the presence of theantibody/antigen complex by histoenzymology using a chromogen substrate.With suitable amplification, a conjugate can be produced which ismacroscopically visible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousforms of hyperproliferative diseases, such as cancer, includingleukemia, for example. Here, a biological and/or clinical samplesuspected of containing a wild type or mutant protein, polypeptide,peptide and/or mutant is used. However, these embodiments also haveapplications to non-clinical samples, such as in the titering of antigenor antibody samples, for example in the selection of hybridomas.

H. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, the antibodies of the invention are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the wild type and/or mutant protein antigen, such as aclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound wild typeand/or mutant protein antigen may be detected. Detection is generallyachieved by the addition of another antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection may also be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing the wildtype and/or mutant protein antigen are immobilized onto the well surfaceand/or then contacted with the antibodies of the invention. Afterbinding and/or washing to remove non-specifically bound immunecomplexes, the bound antibodies are detected. Where the initialantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which the wild type and/or mutant proteins,polypeptides and/or peptides are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst wild type or mutant protein are added to the wells, allowed tobind, and/or detected by means of their label. The amount of wild typeor mutant protein antigen in an unknown sample is then determined bymixing the sample with the labeled antibodies against wild type and/ormutant before and/or during incubation with coated wells. The presenceof wild type and/or mutant protein in the sample acts to reduce theamount of antibody against wild type or mutant protein available forbinding to the well and thus reduces the ultimate signal. This is alsoappropriate for detecting antibodies against wild type or mutant proteinin an unknown sample, where the unlabeled antibodies bind to theantigen-coated wells and also reduces the amount of antigen available tobind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/TWEEN. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/TWEEN, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS TWEEN).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

I. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and/or is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in 70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

J. Immunoelectron Microscopy

The antibodies of the present invention may also be used in conjunctionwith electron microscopy to identify intracellular tissue components.Briefly, an electron-dense label is conjugated directly or indirectly tothe antibody. Examples of electron-dense labels according to theinvention are ferritin and gold. The electron-dense label absorbselectrons and can be visualized by the electron microscope.

K. Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. As the antibodies are generally used to detect wild type and/ormutant proteins, polypeptides and/or peptides, the antibodies willpreferably be included in the kit. However, kits including both suchcomponents may be provided. The immunodetection kits will thus comprise,in suitable container means, a first antibody that binds to a wild typeand/or mutant protein, polypeptide and/or peptide, and/or optionally, animmunodetection reagent and/or further optionally, a wild type and/ormutant protein, polypeptide and/or peptide.

In preferred embodiments, monoclonal antibodies will be used. In certainembodiments, the first antibody that binds to the wild type and/ormutant protein, polypeptide and/or peptide may be pre-bound to a solidsupport, such as a column matrix and/or well of a microtitre plate.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with and/orlinked to the given antibody. Detectable labels that are associated withand/or attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and/or all such labels may beemployed in connection with the present invention.

The kits may further comprise a suitably aliquoted composition of thewild type and/or mutant protein, polypeptide and/or polypeptide, whetherlabeled and/or unlabeled, as may be used to prepare a standard curve fora detection assay. The kits may contain antibody-label conjugates eitherin fully conjugated form, in the form of intermediates, and/or asseparate moieties to be conjugated by the user of the kit. Thecomponents of the kits may be packaged either in aqueous media and/or inlyophilized form.

The container means of the kits will be suitable housed and willgenerally include at least one vial, test tube, flask, bottle, syringeand/or other container means, into which the antibody may be placed,and/or preferably, suitably aliquoted. Where wild type and/or mutantVLPT protein, polypeptide and/or peptide, and/or a second and/or thirdbinding ligand and/or additional component is provided, the kit willalso generally contain a second, third and/or other additional containerinto which this ligand and/or component may be placed. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and/or any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionand/or blow-molded plastic containers into which the desired vials areretained.

VIII. Pharmaceutical Preparations

It is also contemplated that pharmaceutical compositions may be preparedusing the novel compositions of the present invention. In such a case,the pharmaceutical composition comprises the novel active composition ofthe present invention and a pharmaceutically acceptable carrier. Aperson having ordinary skill in this art would readily be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of the active component of the presentinvention.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a subject. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In general, a pharmaceutical composition of the present invention maycomprise an E. chaffeensi VLPT polypeptide, polynucleotide, or antibodyand/or mixtures thereof.

A protein may be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids such as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents that target a polypeptide or thesecretion thereof or additional agent dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical,”“pharmaceutically acceptable,” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one agent that targetsthe polypeptide or the secretion thereof and/or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The invention may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The invention may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the composition is prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations that are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

IX. Exemplary Kits of the Invention

In particular embodiments of the invention, there is a kit housed in asuitable container. The kit may be suitable for diagnosis, treatment,and/or protection for an individual from Ehrlichia, such as Ehrlichiachaffeensis. In particular embodiments, the kit comprises in a suitablecontainer an agent that targets an E. chaffeensis VLPT antigen. Theagent may be an antibody, a small molecule, a polynucleotide, apolypeptide, a peptide, or a mixture thereof. The agent may be providedin the kit in a suitable form, such as sterile, lyophilized, or both,for example. In particular embodiments, the kit comprises an antibodyagainst one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:15;and/or related proteins thereof. Other E. chaffeensis VLPT-relatedimmunogenic-related compositions (including polypeptides, peptides, orantibodies) not specifically presented herein may also be included.

The kit may further comprise one or more apparatuses for delivery of acomposition to an individual in need thereof. The apparatuses mayinclude a syringe, eye dropper, needle, biopsy tool, scoopula, catheter,and so forth, for example.

In embodiments wherein the kit is employed for a diagnostic purpose, thekit may further provide one or more detection compositions and/orapparatuses for identifying an E. chaffeensis VLPT antigen. Such anembodiment may employ a detectable label, such as for an antibody, forexample, and the label may be fluorescent, radioactive,chemiluminescent, or colorimetric, for example.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Materials and Methods

Culture and Purification of Ehrlichiae.

E. canis (Jake strain) and E. chaffeensis (Arkansas strain) werepropagated as previously described (McBride et al., 2001). Ehrlichiaewere purified by size exclusion chromatography over Sephacryl S-1000(Amersham Biosciences, Piscataway, N.J.) as previously described(Rikihisa et al., 1992). The fractions containing bacteria were frozenand utilized as antigen and DNA sources.

Preparation of E. Chaffeensis Genomic DNA and Antigen.

Genomic DNA and antigen were purified from E. chaffeensis (Arkansasstrain) as previously described (McBride et al., 1996).

PCR Amplification of the E. chaffeensis VLPT Gene Fragments.

Oligonucleotide primers for the amplification of the E. chaffeensis VLPTgene fragments were designed manually or by using Primer Select(Lasergene v5.08, DNAStar, Madison, Wis.) according to the sequence inGenBank (accession number AF121232) and synthesized (Sigma-Genosys,Woodlands, Tex.) (Table 2). Seven gene fragments corresponding to thefour single TRs (VLPT-R4, VLPT-R3, VLPT-R2, and VLPT-R1), the C-terminus(VLPT-C), the combination of repeats R3 and R2 (VLPT-R32), and thenearly full-length VLPT (VLPT-R4321-C) containing multiple repeats (R4,R3, R2, and R1) and C-terminus of E. chaffeensis VLPT gene wereamplified using a PCR HotMaster Mix (Eppendorf, Westbury, N.Y.) and E.chaffeensis (Arkansas strain) genomic DNA as the template (Tables 2 and3). The thermal cycling profile was: 95° C. for 4 min, 35 cycles of 94°C. for 30 s, annealing temperature (3° C. less than the lowest primerT_(m)) for 30 s, and 72° C. for the appropriate extension time (30 s/500base pairs) followed by a 72° C. extension for 7 min and a 4° C. hold.

Expression and Purification of the Recombinant E. chaffeensis VLPTProteins.

The amplified PCR products were cloned directly into the pBAD/Thio-TOPO(Invitrogen, Carlsbad, Calif.) or pTriEx-6 3C/LIC expression vector(Novagen, Madison, Wis.). Escherichia coli cells (TOP10; Invitrogen)were transformed with the plasmid containing the E. chaffeensis VLPTgene fragments, and positive transformants were screened by PCR for thepresence of the insert and proper orientation, and were sequenced withan ABI Prism 377XL DNA sequencer (Applied Biosystems, Foster City,Calif.) at the University of Texas Medical Branch Protein Chemistry CoreLaboratory. Recombinant protein expression was performed for 4 h afterinduction with 0.2% arabinose (pBAD/Thio-TOPO) or 0.5 mMisopropyl-β-D-thiogalactopyranoside (IPTG; pTriEx-6 3C/LIC). Recombinantproteins were purified under native conditions using HisSelect® columns(for pBAD/Thio-TOPO; Sigma, St. Louis, Mo.) or Strep•Tactin® Superflowcolumns (for pTriEx-6 3C/LIC; Novagen) and quantitated with the BCAprotein assay (Pierce, Rockford, Ill.) according to the manufacturers'instructions.

E. chaffeensis VLPT Synthetic Peptides.

Five synthetic peptides corresponding to the N-terminal fragment(VLPT-N; 17 amino acids) and four individual TR units (R4, R3, R2, andR1; 30 amino acids each) of E. chaffeensis VLPT protein as well as sevenoverlapping peptides corresponding to the different regions of R3 (R3-1to R3-7) and a 20-amino-acid N-terminal peptide of R4 (R4-N) weresynthesized (Bio-Synthesis, Lewisville, Tex.) (Table 3). The lyophilizedpowder was resuspended in molecular biology grade water (1 mg/ml).

TABLE 2 E chaffeensis (Arkansas) genomic coding sequenceand protein sequence; both available under GenBank®Accession AF121232, incorporated by reference herein SEQ ID Sequence NO:MSQFSEDNMGNIQMPFDSDSHEPSHLELPSLSEEVIQLESD 1LQQSSNSDLHGSFSVELFDPFKEAVQLGNDLQQSSDSDLHGSFSVELFDPSKEEVQLESDLQQSSNSDLHESSFVELPGPSKEEVQFEDDAKNVVYGQDHVSLSELGLLLGGVFSTMNYLSGYTPYYYHHYCCYNPYYYFDYVTPDYCHHCSESSLEtttatatttatatatgattaatatataatgataatggtatg 16tggttataactgcttattagttgatcatgtacctgtgtgttatgttaaatagggtataaatatgtcacaattctctgaagataatatgggtaatatacaaatgccttttgattctgattcacatgagccttctcatcttgagctacctagtctttctgaagaagtgattcaattagagagtgatctacaacaatcttctaattctgatttacacgggtctttttctgttgagttatttgatccttttaaagaagcagttcaattggggaatgatctacaacaatcttctgattctgatttacacgggtctttttctgttgagttatttgatccttctaaagaagaagttcaattggagagtgatctacaacaatcttctaattctgatttacacgagtcttcttttgttgagttacctggtccttccaaagaagaagttcaattcgaagatgatgctaaaaatgtagtatatggacaagaccatgttagtttatctgaattaggcttattgttaggtggtgtttttagtacaatgaattatttgtctggttatacaccgtattattatcatcattattgttgttataatccttattattattttgattatgttactccagattattgtcatcactgtagtgaaagtagtttagagtaggatatttagaaatataaatggttgttgacttcacaaaaggtgtagttttatatgttttatgctgttttatagtgttataa ggatatgagttgtttttactattttt

Antisera.

A convalescent anti-E. chaffeensis dog serum was derived fromexperimentally infected dog (no. 2251). Sera from HME patients were akind gift from Focus Technologies (Cypress, Calif.). Rabbit anti-VLPT-R3antiserum was generated against the synthetic E. chaffeensisVLPT-R3KLH-conjugated peptide by a commercial vendor (Bio-Synthesis).

Gel Electrophoresis and Western Immunoblotting.

Purified E. chaffeensis or E. canis whole-cell lysates or recombinantproteins were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to nitrocellulose, andWestern immunoblotting performed as previously described (McBride etal., 2003), except that primary dog and human sera were diluted 1:100and rabbit anti-VLPT-R3 antiserum was diluted 1:2,000.

Carbohydrate Detection.

Glycan detection on the recombinant protein VLPT was performed with adigoxigenin glycan detection kit (Roche, Indianapolis, Ind.) aspreviously described (McBride et al., 2000).

ELISA. Enzyme-linked immunosorbent assay (ELISA) plates (MaxiSorp; NUNC,Roskilde, Denmark) were coated (0.5 μg/well; 50 μl) with recombinantproteins or synthetic peptides in phosphate-buffered saline (pH 7.4).Proteins and peptides were adsorbed to the ELISA plates overnight at 4°C. with gentle agitation, subsequently washed thrice with 200 μlTris-buffered saline containing 0.2% TWEEN 20 (TBST) and blocked with100 μl 3% bovine serum albumin (BSA) in TBST for 1 h at room temperaturewith agitation, and washed again. Convalescent anti-E. chaffeensis dogor human sera diluted (1:100) in 3% BSA-TBST were added to each well (50μl) and incubated at room temperature for 1 h with gentle agitation. Theplates were washed four times, and 50 μl alkaline phosphatase-labeledgoat anti-dog or human IgG (H+L) secondary antibody (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) diluted (1:5,000) in 3% BSA-TBST wasadded and incubated for 1 h at room temperature. The plates were washedfour times, and substrate (100 μl, BluePhos; Kirkegaard & PerryLaboratories) was added to each well. The plates were incubated for 30min in the dark with agitation, and color development was read on amicroplate reader (VersaMax; Molecular Devices, Sunnyvale, Calif.) atA₆₅₀, and data were analyzed by SoftmaxPro v4.0 (Molecular Devices).Optical density (OD) readings represent the means for three wells(.+-.standard deviations) with the OD of the buffer-only wellssubtracted.

Immunoelectron Microscopy.

Immunogold electron microscopy was performed on E. chaffeensis-infectedDH82 cells as previously described (McBride et al. 2005), except thatprimary rabbit anti-VLPT-R3 peptide serum was diluted 1:10,000.Uninfected DH82 cells were used as a negative control.

Mass Spectrometry.

Mass spectrometry was performed using matrix-assisted laserdesorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer(MS) (Voyager-DE STR; Applied Biosystems) at the University of TexasMedical Branch Mass Spectrometry Core Laboratory.

Analysis of Secreted VLPT Protein.

E. chaffeensis-infected DH82 cell culture supernatants (1 ml) werecollected everyday without disturbing the cell monolayer and centrifugedat high speed (10,000×g for 5 min) to pellet cells and bacteria.Supernatants were subsequently concentrated 10-fold (Centricon ultracentrifugal filter, 10-kDa cutoff; Millipore, Billerica, Mass.) for gelelectrophoresis and Western immunoblotting using anti-VLPT-R3 specificpolyclonal antibody.

Sequence Analysis.

The E. chaffeensis VLPT was evaluated for potential O-linkedglycosylation and phosphorylation with the computational algorithms ofthe YinOYang v1.2 program by Julenius et al., 2005, and NetPhos v2.0program by Blom et al., 1999. Potential signal sequence or non-classicalsecretion was identified with the computational algorithms of theSignalP 3.0 and SecretomeP 2.0 programs by Bendtsen et al., 2004,trained on gram-negative bacteria. Nucleic acid and amino acidalignments were performed with MegAlign (Lasergene v5.08, DNAStar). E.chaffeensis VLPT epitopes were examined for homology to other Ehrlichiaspp. proteins (including VLPT orthologs) using the protein-protein BasicLocal Alignment Search Tool (BLAST).

TABLE 3 Exemplary E. chaffeensis VLPT synthetic polypeptides AminoSEQ ID Peptide Sequence acids NO: N MSQFSEDNMGNIQMPFD 17 2 R4SDSHEPSHLELPSLSEEVIQLESDLQQSSN 30 3 R3 SDLHGSFSVELFDPFKEAVQLGNDLQQSSD 304 R2 SDLHGSFSVELFDPSKEEVQLESDLQQSSN 30 5 R1SDLHESSFVELPGPSKEEVQFEDDAKNVVY 30 6 R3-1 SDLHGSFSVELFDP 14 7 R3-2SDLHGSFSVELFDPFKE 17 8 R3-3 HGSFSVELFDPFKE 14 9 R3-4 HGSFSVELFDPFKEAVQ17 10 R3-5 HGSFSVELFDPFKEAVQLGN 20 11 R3-6 VELFDPFKEAVQLGND 16 12 R3-7FKEAVQLGNDLQQSSD 16 13 R4-N HEPSHLELPSLSEEVIQLES 20 14 CGQDHVSLSELGLLLGGVFSTMNYLSGYTPY 61 15 YYHHYCCYNPYYYFDYVTPDYCHHCSESSL E

TABLE 4 Oligonucleotide primers for amplification of theE. chaffeensis VLPT gene fragments Forward Reverse Amplicon FragmentPrimer Sequence Primer Sequence Size R4 4F TCTGATTCACATGAGCCTTC 4(2)RATTAGAAGATTGTTGTAGATCAC 90 (SEQ ID NO: 17) TC (SEQ ID NO: 18) R3 3(2)FTCTGATTTACACGGGTCTT 3R ATCAGAAGATTGTTGTAGATCAT 90 (SEQ ID NO: 19)(SEQ ID NO: 20) R2 3(2)F TCTGATTTACACGGGTCTT 4(2)RATTAGAAGATTGTTGTAGATCAC 90 (SEQ ID NO: 21) TC (SEQ ID NO: 22) R1 1FTCTGATTTACACGAGTCTTCT 1R ATATACTACATTTTTAGCATCAT 90 (SEQ ID NO: 23) CTTC(SEQ ID NO: 24) C CF GGACAAGACCATGTTAGTTT CR CTCTAAACTACTTTCACTACAGT 183(SEQ ID NO: 25) G (SEQ ID NO: 26) R4321-C 4F TCTGATTCACATGAGCCTTC CRCTCTAAACTACTTTCACTACAGT 543 (SEQ ID NO: 27) G (SEQ ID NO: 28) R32 3(2)FTCTGATTTACACGGGTCTT 4(2)R ATTAGAAGATTGTTGTAGATCAC 180 (SEQ ID NO: 29) TC(SEQ ID NO: 30) R32a 3F-lic CAGGGACCCGGTTCTTCTAAT 2R-licGGCACCAGAGCGTTTTAATTAGA 213 TCTGATTTACACGG AGATTGTTGTAGATCACTC(SEQ ID NO: 31) (SEQ ID NO: 32)

Example 2 Characterization of the E. chaffeensis VLPT Protein

E. chaffeensis VLPT Protein Composition and Characteristics.

Serine (33 residues; 16.7%), leucine (22; 11.1%), glutamate (20; 10.1%),and aspartate (17; 8.6%) were the most frequently occurring amino acidsin the E. chaffeensis VLPT protein, accounting for 46.5% of the entireamino acid content (FIG. 1A). Moreover in the repeat region of VLPTprotein, the occurrences of these four residues became more frequentwith a composition of 20%, 12.5%, 13.3% and 10%, respectively, whichaccounted for 55.8% of the entire repeat region amino acid content. Fourcysteine residues associated with disulfide bonds were present in thecarboxyl-terminal domain of the protein, but this domain was dominatedby tyrosine residues (19.7%). Due to the large proportion of stronglyacidic residues glutamate and aspartate, the VLPT protein was highlyacidic (pI 3.8). The N-terminal region (17 amino acids) and the largestdomain, the TR region (120 amino acids), were highly acidic (pI 3.2 and3.8, respectively), and carboxyl-terminal domain (61 amino acids) theleast acidic (pI 4.7).

The TRs of VLPT were non-identical, but R3 and R2 had the highest aminoacid identity (83%) and R4 and R1 had 53% and 49% identity with R3,respectively (FIG. 1B). A BLAST search with amino acid sequences fromVLPT-R3 and VLPT-R4 found no homology between the VLPT repeats and otherknown ehrlichial proteins or proteins from organisms in closely relatedgenera.

Identification of Native VLPT Protein.

Western blotting identified a native protein with a molecular mass of˜32 kDa (˜6.2 kDa larger than predicted mass of 25.8 kDa) and five lessprominent proteins (22-30 kDa) in E. chaffeensis whole-cell lysates andsupernatants from E. chaffeensis-infected cells that reacted withmonospecific rabbit antiserum against the synthetic VLPT-R3 peptide(FIG. 2A). Furthermore, anti-VLPT-R3 antibody did not react with E.canis whole cell lysate (FIG. 2A). A protein of similar size (˜32 kDa)in E. chaffeensis whole cell lysates and supernatants from infectedcultures also reacted with anti-E. chaffeensis dog serum (FIG. 2B).Pre-immunization rabbit serum or dog serum controls did not recognize E.chaffeensis whole cell lysates or supernatants (data not shown).

Epitope Containing Regions of VLPT.

To determine the major epitope-containing regions of VLPT, recombinantproteins corresponding to distinct VLPT domains (R4321-C, R32, R1, R2,R3 R4, and C; FIG. 3) were expressed. All of the recombinant VLPTproteins expressed in pBAD/Thio-TOPO exhibited molecular masses thatwere substantially larger by SDS-PAGE than that predicted by amino acidsequence, except for VLPT-R3 and VLPT-C. The VLPT-R32 expressed in analternative vector pTriEx6 3C/LIC with substantially smaller N-terminalfusion protein (2.4 kDa compared to 13.1 kDa for pBAD/Thio) alsoexhibited an increased molecular mass (3.7 kDa larger than predicted),but smaller than that of VLPT-R32 expressed in pBAD/Thio (5.2 kDa largerthan predicted). The increased molecular mass exhibited by recombinantVLPT proteins indicated the protein was posttranslationally modified;moreover, several predicted glycosylation sites were identified on VLPTby computational algorithm. However, carbohydrate was not detected onany recombinant E. chaffeensis VLPT polypeptides (data not shown). Tofurther confirm the actual molecular mass of one recombinant VLPTprotein, the mass of the two-repeat containing VLPT-R32 expressed inpTriEx6 3C/LIC was determined by MALDI-TOF. The mass of VLPT-R32 was9,206 Da, slightly smaller than the mass (9,325 Da) predicted by theamino acid sequence (excluding the 2.4 kDa expression tag),demonstrating post translational modifications were not present.

By Western immunoblot, the large TR-containing proteins R4321-C and R32and individual repeat units R2 and R3 reacted strongly with anti-E.chaffeensis dog serum, but recombinant fragments representing thecarboxy terminal domain (C) and repeat units R1 and R4 were not reactivewith anti-E. chaffeensis dog serum (FIG. 4A). Reactivity of the VLPT(synthetic polypeptides and corresponding recombinant proteins) withanti-E. chaffeensis dog serum was also examined by ELISA to identifypotential conformational epitopes (FIG. 4B). VLPT-N (synthetic), VLPT-C(recombinant), or VLPT-R1 (synthetic and recombinant) polypeptides didnot react with anti-E. chaffeensis dog serum. Similar to results byWestern immunoblotting, VLPT-R3 or R2 peptides (synthetic andrecombinant) reacted strongly with anti-E. chaffeensis dog serum;however, the VLPT-R3 (synthetic) was more immunoreactive than VLPT-R2(synthetic). Conversely, the recombinant VLPT-R4, which was notimmunoreactive by Western immunoblot, reacted (synthetic andrecombinant) strongly with anti-E. chaffeensis dog serum by ELISA,indicating that a conformational epitope was present in VLPT-R4 (FIGS.4A and B).

Identification of major VLPT immunodeterminants. To further localize themajor epitopes of E. chaffeensis VLPT protein, seven overlappingpeptides (designated R3-1 to R3-7) corresponding to the differentlocations within VLPT-R3 were reacted with anti-E. chaffeensis dog serum(FIGS. 3 and 5A). Peptides R3-6 and R3-7 (C-terminal region) were notimmunoreactive, but R3-2, R3-3, R3-4 and R3-5 corresponding to theN-terminal region were found to react similarly and strongly withanti-E. chaffeensis dog serum by ELISA (FIG. 4B), indicating theN-terminal region 23 amino acids of VLPT-R3 contained a major antibodyepitope. Peptide R3-3 (14 amino acids; HGSFSVELFDPFKE; SEQ ID NO:9) wasthe smallest peptide that reacted strongly with anti-E. chaffeensis dogserum (FIGS. 5A and B). Peptides R3-1 and R3-6, which differed by three(C-terminal) and five amino acids (N-terminal), respectively, were notreactive (FIGS. 5A and B).

To examine and compare the immunodeterminant in VLPT-R4, a 20-amino-acidpeptide (HEPSHLELPSLSEEVIQLES; SEQ ID NO:14) corresponding to the R3-5in VLPT-R3 (FIG. 4A) was not immunoreactive with either dog serum orpatient sera (data not shown), indicating that the third epitope of VLPTin R4 was molecularly distinct, and is consistent with the divergencenoted in the amino acid sequences of R4 compared to R3 and R2 (FIG. 1B).

Immunoreactivity of VLPT-R3 Peptides with HME Patient Sera.

Three HME patient sera (nos. 1, 4 and 12) that had detectable E.chaffeensis antibodies by immunofluorescence assay (IFA) were used toexamine the immunoreactivity of VLPT-R4, R3, and R2 (synthetic andrecombinant) by ELISA (FIG. 6A-C, respectively). Consistent with theimmunoreactivity exhibited with anti-E. chaffeensis dog serum, VLPT-R3and R2 also exhibited the strongest immunoreactivity with HME patientsera, and two patients (nos. 1 and 12) exhibited a strong antibodyresponse to VLPT-R4 (FIGS. 6A-C).

The immunoreactivity of the three HME patient sera with the sevenoverlapping synthetic peptides (R3-1 to R3-7) from VLPT-R3 wasdetermined by ELISA (FIGS. 6A-C). Peptides R3-4 (17 amino acids) andR3-5 (20 amino acids) which contained similar amino acid sequences (seeFIG. 5A) reacted strongly and consistently with the all HME patient seratested (FIGS. 6A-C). Comparing the overlapping peptides, the minimumpeptide sequence critical for this immunodeterminant was 17 amino acids(peptide R3-5). Antibodies from HME patients and the dog experimentallyinfected with E. chaffeensis reacted similarly to VLPT-R3 (FIGS. 4B and6A-C). However, antibodies in human sera were directed primarily againstpeptides R3-4 and R3-5 within VLPT-R3 (FIGS. 6A-C). Normal human serumdid not recognize these peptides and proteins (data not shown).

The reactivity of VLPT-R3 with a larger panel of HME patient sera (14patients) that had detectable E. chaffeensis antibodies was determined.All patient sera reacted with VLPT-R3 (synthetic) (FIG. 6D), indicatingthat this epitope is consistently recognized by humans and thereactivity of antibodies in patient sera with this epitope completelycorrelated with IFA. The normal human serum did not recognize VLPT-R3(FIG. 6D, lane 16)

Temporal Secretion of E. chaffeensis VLPT.

VLPT was detected in supernatants from infected cells as early as 1 daypost infection and increased in quantity through 6 days post infection(FIG. 7). The VLPT protein was not observed in uninfected DH82 cellculture supernatant.

Cellular and Extracellular Localization of VLPT.

Several characterized ehrlichial proteins are differentially expressedon dense-cored ehrlichiae (gp120, gp36, and gp47). However, like itsortholog gp19 of E. canis, the E. chaffeensis VLPT protein was observedon the membrane of morula and surface of both reticulate and dense-coredehrlichiae, but was also detected on the morula fibrillar matrix byimmunoelectron microscopy (FIG. 8A). Anti-VLPT-R3 antibody did not reactwith uninfected DH82 cells (FIG. 8B).

Example 3 Significance of the Present Invention

The initial description of the E. chaffeensis VLPT gene focused on theapplications of the gene for molecular diagnostics and epidemiology.Hence, the VLPT gene has been frequently utilized to differentiateisolates based on differences in the number of TRs units and sequencevariation present in the gene (Sumner et al., 1999; Yabsley et al.,2003). Although a previous study demonstrated that recombinant VLPTreacted with antibodies in HME patient sera, the immunologic propertiesof the VLPT protein were not fully defined (Popov et al., 2000).Notably, the VLPT protein has never been conclusively identified in E.chaffeensis native whole cell lysates, and major immunoreactive proteinscorresponding to its reported molecular mass of 44-kDa (double thepredicted size) have never been identified. Hence, the identity of VLPTand extent of the host response directed against it have remainedundetermined. Recently, it was described that the identification andcharacterization of a conserved, strongly acidic major immunoreactive19-kDa protein (gp19) in E. canis that elicits an early antibodyresponse (McBride et al., 2003). It was also concluded based on genomicand protein analysis that the E. chaffeensis VLPT protein was theortholog of gp19. The role of E. chaffeensis VLPT protein in ehrlichialpathobiology is also unknown, and its lack of relationship with otherknown bacterial proteins provides no clues regarding its potentialfunction. A remarkable feature of VLPT and E. canis gp19 is thehomologous carboxy-terminal domain dominated by tyrosine, indicatingthat it is a functionally important conserved domain.

The discrepancy in the apparent molecular mass of the E. chaffeensisVLPT protein (Arkansas strain) observed in the invention (˜32 kDa) andthat of the recombinant VLPT (˜44 kDa) reported previously was noted,and it is acknowledged that the native VLPT was never identified in aprevious study. Nevertheless, the native VLPT protein (˜32 kDa)identified from the ehrlichial lysate by anti-VLPT-R3 antibody, and themass of the recombinant VLPT protein (without fusion tag) were inagreement. Hence, the evidence generated by the invention has indicatedthat the mass of the VLPT (recombinant and native) is ˜32-kDa, which islarger than the predicted mass (25.7-kDa) but substantially smaller thanpreviously reported (Sumner et al., 1999).

Four pairs (gp200s, gp120/gp140, gp47/gp36, and VLPT/gp19) of majorimmunoreactive protein orthologs in E. chaffeensis and E. canis havebeen identified. Two ortholog pairs are TR-containing proteins, and theVLPT/gp19 also appears to be similar. Although the E. canis gp19 lacksmultiple repeats found in the E. chaffeensis VLPT, it has aSer/Thr/Glu-rich patch that is similar in size and composition to thatof a single serine-rich repeat unit of VLPT, and the majorimmunodeterminant of the gp19 was mapped to the STE-rich patch.Similarly, antibody epitopes have been identified in other serine-richTR-containing ehrlichial protein orthologs including gp36/47 andgp120/140 (Doyle et al., 2006; Yu et al. 1996).

Except for p28/p30, all of the major immunoreactive proteins of E.chaffeensis and E. canis that have been characterized are highly acidicdue to a predominance of glutamate and aspartate, but they also have alarge proportion of polar amino acids, such as serine, which are presentin higher frequency within TRs found in these proteins. Moreover, majorantibody epitopes of these proteins have been mapped to theseserine-rich acidic TRs or acidic domains (Doyle et al., 2006; McBride etal., 2003; McBride et al., 2000; Yu et al., 1997; Yu et al., 2000;Nethery et al., 2007). The amino acid composition of E. canis gp19consisted predominately of three amino acids, serine, glutamate andaspartate. Consistent with other major immunoreactive proteins includinggp19, VLPT has similar predominance of serine, glutamate and aspartatethat are more pronounced in the TRs region. The high frequency of thepolar and acidic amino indicates a direct relationship between the hostimmune response and acidic serine-rich repetitive sequences and domains.

Previously, it was reported that detection of carbohydrate onrecombinant ehrlichial TR-containing proteins that exhibited larger thanpredicted masses similar to their native counterparts. Furthermore, VLPThas been reported to exhibit a larger than predicted mass by gelelectrophoresis, a finding that was also observed in the invention withboth native and recombinant VLPT proteins (Sumner et al., 1999). Thus,the possibility that glycosylation was responsible for this differencewas considered. Serine and threonine residues are linkage sites forO-glycans and some of these amino acids were predicted to be glycanattachment sites on the VLPT. However, unlike other ehrlichial proteins,carbohydrate on the VLPT was not detected, and the mass (as determinedby MALDI-TOF) of a recombinant two repeat containing fragment (VLPT-R32)was consistent with its predicted mass confirming that the abnormalmigration was not due to post-translational modification of VLPT tandemrepeats. In one embodiment, the increase in electrophoretic mobility isbecause VLPT is a highly acidic protein. Others have reported thathighly acidic proteins such as ribonuclease U2 and caldesmon exhibitanomalous electrophoretic behavior that could be normalized afterneutralization (Garcia-Ortega et al., 2005; Graceffa et al., 1992;Moussa et al., 2004). In specific embodiments of the invention, the highacidic amino acid content and low overall pI (3.8) of VLPT explains itselectrophoretic behavior and contributes to the anomalous behavior ofother highly acidic TR-containing ehrlichial proteins.

Three major epitope-containing regions were identified in E. chaffeensisVLPT protein in the non-identical serine-rich repeat units R2, R3 andR4, respectively, which is consistent with the location of epitopes inother ehrlichial TR-containing proteins (Doyle et al., 2006; McBride etal., 2003; McBride et al., 2000). The antibody epitope in R3, whichexhibited the strongest antibody reactivity with both human sera, waslocalized to a 17 amino acid N-terminal region that was highlyhomologous with R2 (two amino acid changes). Thus, antibodies directedagainst R3 would likely cross react with R2. Therefore, the R3 epitopeappears to be the primary immunodeterminant for both human and doganti-VLPT antibodies. Interestingly, the R3 immunodeterminant appearedto be highly dependent on three terminal amino acids (AVQ) in peptideR3-4 when detected by human antibodies, whereas the antibodies reactivein canine serum appeared to be more dependent on three amino acids (FKE)directly upstream. Thus, R3-3 was the minimum epitope sequence (14 aminoacids) for recognition by dog antibodies, and R3-4 (17 amino acids)containing three additional C-terminal amino acids is essential forantibody reactivity with human sera. Interestingly, R4 was the mostdivergent repeat, and was not reactive by Western immunoblotting but wasreactive with antibody in ELISA. This indicates that a distinctconformational epitope was present in R4. Conformational epitopes havebeen described in Ehrlichia and Anaplasma species (Chen et al., 1996;Munodzana et al., 1998). Thus, R4 contributes to the immunoreactivity ofVLPT independent of R3. The smaller R4 peptide (20 amino acids) thatcorresponds to R3-5 in VLPT-R3 was not immunoreactive; however, the fullrepeat (30 amino acids) was immunoreactive, which supports theconclusion that this epitope is discontinuous and requires the entirerepeat sequence to create the epitope.

The epitopes identified in VLPT repeat units appear to bespecies-specific, as the anti-VLPT-R3 antibody did not cross-react withclosely related E. canis and amino acid homology was not observedbetween VLPT-R2, R3 and R4, and proteins of other Ehrlichia species orclosely related pathogens. This is consistent with the previouslyreported antibody epitope identified in E. canis gp19 (VLPT ortholog),which was also species-specific (McBride et al., 2006). Furthermore,similar species-specific epitopes in E. chaffeensis and E. canis proteinorthologs including the gp120/gp140, gp47/gp36, gp200s have beenidentified (Doyle et al., 2006; McBride et al., 2003; McBride et al.,2000; Yu et al., 1997; Yu et al, 2000). The current findings furthersupport the embodiment that antibodies generated against E. chaffeensisare directed primarily at species-specific epitopes. Hence, antibodiesgenerated against one Ehrlichia species may provide little or noprotection against a closely related pathogen, such as E. canis in thiscase. However, species-specific antigens such as VLPT are excellentcandidates for the development of sensitive species-specificimmunodiagnostics and are useful for epidemiologic studies.

There is evidence that ehrlichial TR-containing proteins such as E.chaffeensis gp120 and gp47 are secreted (Doyle et al., 2006; Popov etal., 2000). In the invention, it is demonstrated that the VLPT proteinis also secreted. The mechanism of secretion appears to besec-independent because VLPT does not have an amino-terminal signalsequence. VLPT was predicted by SecretomeP 2.0 to be secreted by anonclassical and leaderless secretion system; therefore, secretion ofVLPT and other TR-containing proteins may occur by a similar mechanism,including E. chaffeensis gp120 and gp47, which also lack an N-terminalsignal sequence, but are found outside the bacterium in the morula andin the infected cell culture supernatants. Genes encoding type IVsecretion system components have been reported in both Ehrlichia andAnaplasma (Dunning Hotopp et al., 2006; Ohashi et al., 2002), and AnkAof A. phagocytophilum appears to be secreted by this system (Lin et al.,2007). However, the VLPT does not appear to contain a type IV effectorprotein consensus sequence and could be a substrate of other secretionsystems (sec-dependent and sec-independent) that have been identified inEhrlichia species (Dunning Hotopp et al., 2006).

Distinct from the differential expression (on the dense-coredehrlichiae) of E. chaffeensis gp120 and gp47, but consistent with thelocalization of E. canis gp19 (Doyle et al., 2006; Popov et al., 2000),E. chaffeensis VLPT protein was detected on both morphologic forms,reticulate and dense-cored ehrlichiae, but was primarily foundextracellularly associated with the morula fibrils and morula membrane.Thus, the VLPT protein does not appear to be a major surface protein andis not associated specifically with the infectious form of ehrlichiae(dense cored). The secretion of VLPT into the morula space and membraneindicates a potentially important role in morula maintenance or as avirulence factor.

The majority of the characterized major immunoreactive proteins ofEhrlichia species are acidic TR-containing proteins that have commonamino acid usage and elicit strong humoral immune responses directed atTRs. The host immune response appears to be primarily directed atepitopes within TRs, which indicates that all of these proteins interactsimilarly with the host immune response. In specific embodiments of theinvention, antibodies directed at specific epitopes in TR-proteins areprotective.

Example 4 Vaccines of the Invention

In particular aspects of the invention, the immunogenic compositions ofthe present invention are suitable as a vaccine, such as a subunitvaccine. In other aspects of the invention, the immunogenic compositionsare referred to as immunoprotective.

Specifically, one or more compositions of the invention, such as thosecomprising an E. chaffeensis VLPT epitope, for example, are administeredto a mammal, such as a human, canine, bovine, or equine animal, forexample. Serum from the mammal may be assayed for an immune response,such as by detecting antibodies in the serum. The mammal is thensubjected to subsequent challenge with the pathogenic organism, such asthe E. canis organism, or another appropriate composition, andimmunoprotection is determined. Controls may be employed, such asimmunization with, for example, a mutated epitope or an epitope thatdoes not comprise a carbohydrate moiety. Complete or partial protectionagainst the subsequent challenge demonstrates the immunoprotectivenature of the composition, and the composition is a vaccine. Partialprotection may be defined as protecting from developing or delaying fromdeveloping at least one symptom of the infection or protecting from atleast one symptom becoming worse.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

Patents and Patent Applications

-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 4,554,101-   PCT/US07/75343

Publications

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Hyg.    54:405-412.-   Collins, N. E., J. Liebenberg, E. P. de Villiers, K. A. Brayton, E.    Louw, A. Pretorius, F. E. Faber, H. H. van, A. Josemans, K. M.    van, H. C. Steyn, M. F. van Strijp, E. Zweygarth, F. Jongejan, J. C.    Maillard, D. Berthier, M. Botha, F. Joubert, C. H. Corton, N. R.    Thomson, M. T. Allsopp, and B. A. Allsopp. 2005. The genome of the    heartwater agent Ehrlichia ruminantium contains multiple tandem    repeats of actively variable copy number. Proc. Natl. Acad. Sci.    U.S.A 102:838-843.-   Doyle, C. K., A. M. Cardenas, D. M. Aguiar, M. B. Labruna, L. M.    Ndip, X. J. Yu, and McBride J. W. 2006. Molecular characterization    of E. canis gp36 and E. chaffeensis gp47 tandem repeats among    different geographic locations. Ann. N.Y. Acad. Sci. 1063.-   Doyle, C. K., K. A. Nethery, V. L. Popov, and J. W. McBride. 2006.    Differentially expressed and secreted major immunoreactive protein    orthologs of Ehrlichia canis and E. chaffeensis elicit early    antibody responses to epitopes on glycosylated tandem repeats.    Infect. Immun. 74:711-720.-   Dunning Hotopp, J. C., M. Lin, R. Madupu, J. Crabtree, S. V.    Angiuoli, J. Eisen, R. Seshadri, Q. Ren, M. Wu, T. R. Utterback, S.    Smith, M. Lewis, H. Khouri, C. Zhang, H. Niu, Q. Lin, N. Ohashi, N.    Zhi, W. Nelson, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, J.    Sundaram, S. C. Daugherty, T. Davidsen, A. S. Durkin, M.    Gwinn, D. H. Haft, J. D. Selengut, S. A. Sullivan, N. Zafar, L.    Zhou, F. Benahmed, H. Forberger, R. Halpin, S. Mulligan, J.    Robinson, O. White, Y. Rikihisa, and H. Tettelin. 2006. Comparative    genomics of emerging human ehrlichiosis agents. PLoS Genet. 2:e21.-   Frutos, R., A. Viari, C. Ferraz, A. Morgat, S. Eychenie, Y.    Kandassamy, I. Chantal, A. Bensaid, E. Coissac, N. Vachiery, J.    Demaille, and D. Martinez. 2006. Comparative genomic analysis of    three strains of Ehrlichia ruminantium reveals an active process of    genome size plasticity. J Bacteriol 188:2533-2542.-   Garcia-Ortega, L., I. R. De, V, A. Martinez-Ruiz, M. Onaderra, J.    Lacadena, P. A. Martinez del, and J. G. Gavilanes. 2005. Anomalous    electrophoretic behavior of a very acidic protein: ribonuclease U2.    Electrophoresis 26:3407-3413.-   Graceffa, P., A. Jancso, and K. Mabuchi. 1992. Modification of    acidic residues normalizes sodium dodecyl sulfate-polyacrylamide gel    electrophoresis of caldesmon and other proteins that migrate    anomalously. Arch. Biochem. Biophys. 297:46-51.-   Johannesson et al., 1999, “Bicyclic tripeptide mimetics with reverse    turn inducing properties.” J. Med. Chem. 42:601-608.-   Julenius, K., A. Molgaard, R. Gupta, and S. Brunak. 2005.    Prediction, conservation analysis, and structural characterization    of mammalian mucin-type O-glycosylation sites. Glycobiology    15:153-164.-   Lin, M., A. den Dulk-Ras, P. J. Hooykaas, and Y. Rikihisa. 2007.    Anaplasma phagocytophilum AnkA secreted by type IV secretion system    is tyrosine phosphorylated by Abl-1 to facilitate infection. Cell    Microbiol. 9:2644-2657.-   Mavromatis, K., C. K. Doyle, A. Lykidis, N. Ivanova, M. P.    Francino, P. Chain, M. Shin, S. Malfatti, F. Larimer, A.    Copeland, J. C. Detter, M. Land, P. M. Richardson, X. J. Yu, D. H.    Walker, J. W. McBride, and N. C. Kyrpides. 2006. The genome of the    obligately intracellular bacterium Ehrlichia canis reveals themes of    complex membrane structure and immune evasion strategies. J    Bacteriol 188:4015-4023.-   McBride J. W., C. K. Doyle, X. F. Zhang, A. M. Cardenas, V. L.    Popov, K. A. Nethery, and M. E. Woods. 2006. Ehrlichia canis 19-kDa    glycoprotein ortholog of E. chaffeensis variable length PCR target    contains a single serine-rich epitope defined by a carbohydrate    immunodeterminant. Infect. Immun.-   McBride J W, R. E. Corstvet, S. D. Gaunt, C. Boudreaux, T. Guedry,    and D. H. Walker. 2003. Kinetics of antibody response to Ehrlichia    canis immunoreactive proteins. Infect. Immun. 71:2516-2524.-   McBride, J. W., J. E. Corner, and D. H. Walker. 2003. Novel    immunoreactive glycoprotein orthologs of Ehrlichia spp. Ann. N.Y.    Acad. Sci. 990:678-684.-   McBride, J. W., L. M. Ndip, V. L. Popov, and D. H. Walker. 2002.    Identification and functional analysis of an immunoreactive    DsbA-like thio-disulfide oxidoreductase of Ehrlichia spp. Infect.    Immun. 70:2700-2703.-   McBride, J. W., R. E. Corstvet, E. B. Breitschwerdt, and D. H.    Walker. 2001. Immunodiagnosis of Ehrlichia canis infection with    recombinant proteins. J. Clin. Microbiol. 39:315-322.-   McBride, J. W., X. J. Yu, and D. H. Walker. 1999. Molecular cloning    of the gene for a conserved major immunoreactive 28-kilodalton    protein of Ehrlichia canis: a potential serodiagnostic antigen.    Clin. Diag. Lab. Immunol. 6:392-399.-   McBride, J. W., X. J. Yu, and D. H. Walker. 2000. Glycosylation of    homologous immunodominant proteins of Ehrlichia chaffeensis and E.    canis. Infect. Immun. 68:13-18.-   McBride, J. W., X. Yu, and D. H. Walker. 2000. A conserved,    transcriptionally active p28 multigene locus of Ehrlichia canis.    Gene 254:245-252.-   Munodzana, D., T. F. McElwain, D. P. Knowles, and G. H.    Palmer. 1998. Conformational dependence of Anaplasma marginale major    surface protein 5 surface-exposed B-cell epitopes. Infection &    Immunity 66:2619-2624.-   Nethery, K. A., C. K. Doyle, B. L. Elsom, N. K. Herzog, V. L. Popov,    and J. W. McBride. 2005. Ankyrin repeat containing immunoreactive    200 kD glycoprotein (gp200) orthologs of Ehrlichia chaffeensis and    Ehrlichia canis are translocated to the nuclei of infected    monocytes, p. O-60. In 4th International Conference on Rickettsiae    and Rickettsial Diseases, Longrono, Spain.-   Nethery, K. A., C. K. Doyle, X. Zhang, and J. W. McBride. 2007.    Ehrlichia canis gp200 contains dominant species-specific antibody    epitopes in terminal acidic domains. Infect. Immun. 75:4900-4908.-   Ohashi, N., N. Zhi, Q. Lin, and Y. Rikihisa. 2002. Characterization    and transcriptional analysis of gene clusters for a type IV    secretion machinery in human granulocytic and monocytic ehrlichiosis    agents. Infect. Immun. 70:2128-2138.-   Paddock, C. D. and J. E. Childs. 2003. Ehrlichia chaffeensis: a    prototypical emerging pathogen. Clin. Microbiol. Rev. 16:37-64.-   Popov, V. L., X. J. Yu, and D. H. Walker. 2000. The 120-kDa outer    membrane protein of Ehrlichia chaffeensis: preferential expression    on dense-core cells and gene expression in Escherichia coli    associated with attachment and entry. Microb. Path. 28:71-80.-   Rikihisa, Y., S. A. Ewing, J. C. Fox, A. G. Siregar, F. H. Pasaribu,    and M. B. Malole. 1992. Analyses of Ehrlichia canis and a canine    granulocytic Ehrlichia infection. J. Clin. Microbiol. 30:143-148.-   Singu, V., H. Liu, C. Cheng, and R. R. Ganta. 2005. Ehrlichia    chaffeensis expresses macrophage- and tick cell-specific    28-kilodalton outer membrane proteins. Infect. Immun. 73:79-87.-   Sumner, J. W., J. E. Childs, and C. D. Paddock. 1999. Molecular    cloning and characterization of the Ehrlichia chaffeensis    variable-length PCR target: an antigen-expressing gene that exhibits    interstrain variation. J. Clin. Microbiol. 37:1447-1453.-   Vita et al., 1998, “Novel miniproteins engineered by the transfer of    active sites to small natural scaffolds.” Biopolymers 47:93-100.-   Weisshoff et al., 1999, “Mimicry of beta II'-turns of proteins in    cyclic pentapeptides with one and without D-amino acids.” Eur. J.    Biochem. 259:776-788.-   Yabsley, M. J., S. E. Little, E. J. Sims, V. G. Dugan, D. E.    Stallknecht, and W. R. Davidson. 2003. Molecular variation in the    variable-length PCR target and 120-kilodalton antigen genes of    Ehrlichia chaffeensis from white-tailed deer (Odocoileus    virginianus). J. Clin. Microbiol. 41:5202-5206.-   Yu, X. J., J. W. McBride, C. M. Diaz, and D. H. Walker. 2000.    Molecular cloning and characterization of the 120-kilodalton protein    gene of Ehrlichia canis and application of the recombinant    120-kilodalton protein for serodiagnosis of canine ehrlichiosis. J.    Clin. Microbiol. 38:369-374.-   Yu, X. J., J. W. McBride, X. F. Zhang, and D. H. Walker. 2000.    Characterization of the complete transcriptionally active Ehrlichia    chaffeensis 28 kDa outer membrane protein multigene family. Gene    248:59-68.-   Yu, X. J., P. A. Crocquet-Valdes, L. C. Cullman, V. L. Popov,    and D. H. Walker. 1999. Comparison of Ehrlichia chaffeensis    recombinant proteins for serologic diagnosis of human monocytotropic    ehrlichiosis. J. Clin. Microbiol. 37:2568-2575.-   Yu, X. J., P. Crocquet-Valdes, and D. H. Walker. 1997. Cloning and    sequencing of the gene for a 120-kDa immunodominant protein of    Ehrlichia chaffeensis. Gene 184:149-154.-   Yu, X. J., P. Crocquet-Valdes, L. C. Cullman, and D. H.    Walker. 1996. The recombinant 120-kilodalton protein of Ehrlichia    chaffeensis, a potential diagnostic tool. J. Clin. Microbiol.    34:2853-2855.-   Yu, X., J. F. Piesman, J. G. Olson, and D. H. Walker. 1997. Short    report: geographic distribution of different genetic types of    Ehrlichia chaffeensis. Am. J. Trop. Med Hyg. 56:679-680.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A polypeptide composition comprising one or moreof the following: (a) an isolated polypeptide that is no more than 50amino acids in length comprising one or more amino acid sequencesselected from the group consisting of: SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11; or (b) an isolatedpolypeptide that is at least 95% identical to the polypeptide of (a);and wherein the one or more isolated polypeptide(s) are bound to a solidsupport.
 2. The polypeptide composition of claim 1, wherein saidisolated polypeptide is dispersed in a pharmaceutically acceptablediluent.
 3. The composition of claim 1, wherein the polypeptide of (a)comprises the amino acid sequence selected from the group consisting of:SEQ ID NO:3 and SEQ ID NO:4.
 4. The composition of claim 1, wherein thepolypeptide of (a) comprises the amino acid sequence selected from thegroup consisting of: SEQ ID NO: 8; SEQ ID NO:9; SEQ ID NO:10; and SEQ IDNO:11.
 5. The composition of claim 1, wherein the polypeptide of (a)comprises the amino acid sequence SEQ ID NO:8.
 6. The composition ofclaim 1, wherein the polypeptide of (a) comprises the amino acidsequence SEQ ID NO:9.
 7. The composition of claim 1, wherein thepolypeptide of (a) comprises the amino acid sequence SEQ ID NO:10. 8.The composition of claim 1, wherein the polypeptide of (a) comprises theamino acid sequence SEQ ID NO:11.
 9. The composition of claim 1, whereinthe polypeptide of (a) comprises the amino acid sequence SEQ ID NO:3.10. The composition of claim 1, wherein the polypeptide of (a) comprisesthe amino acid sequence SEQ ID NO:4.
 11. A kit, housed in a suitablecontainer, comprising the polypeptide composition of claim
 1. 12. Thepolypeptide composition of claim 1, wherein the isolated polypeptideconsists of one or more amino acid sequences selected from the groupconsisting of: SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11.13. The polypeptide composition of claim 1, wherein the isolatedpolypeptide is less than 30 amino acids in length and comprises (a) anamino acid sequence selected from the group consisting of SEQ ID NO:8;SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11; or (b) an isolatedpolypeptide that is at least 95% identical to the polypeptide of (a).14. The polypeptide composition of claim 1, wherein the solid support isa microtiter well, nitrocellulose sheet, immunoblot membrane or columnmatrix.